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Microbial activity is the primary and inevitable factor contributing to seafood spoilage. To assess the spoilage potential of dominant bacteria (Hafnia paralvei and Aeromonas allosaccharophila) isolated from spoiled vacuum-packed grouper fillets under cold storage. In this study, the total viable count (TVC), total volatile basic nitrogen (TVB-N), pH, trichloroacetic acid (TCA)-soluble peptides, water-holding capacity (WHC), nucleotide catabolism, free amino acids (FAAs), and volatile compounds (VOCs) of grouper fillets in vacuum packaging under cold storage with different bacteria inoculated (H. paralvei inoculated, A. allosaccharophila inoculated, and H. paralvei & A. allosaccharophila inoculated) were characterized. The results revealed that H. paralvei exhibited stronger degradation activity, leading to decreased water-holding capacity and increased levels of TVB-N and TCA-soluble peptides. Amino acid analysis showed distinct metabolic patterns for H. paralvei and A. allosaccharophila , with H. paralvei showing a high potential for utilizing FAAs. Both H. paralvei and A. allosaccharophila caused nucleotide catabolism, but A. allosaccharophila produced more hypoxanthine riboside and hypoxanthine. In addition, the analysis of VOCs indicated that H. paralvei produces large content of aldehydes in the middle of storage, particularly benzaldehyde. In addition, the grouper fillets inoculated with H. paralvei & A. allosaccharophila showed similar physicochemical changes to that inoculated with H. paralvei. In conclusion, H. paralvei emerged as the key bacteria responsible for spoilage in vacuum-packed grouper. This study is helpful in further understanding the relationship between spoilage microorganisms and the quality of grouper fillets, thereby providing valuable information for their preservation. [Display omitted] • H. paralvei and A. allosaccharophila dominated the spoilage of chilled vacuum-packed grouper. • H. paralvei had a higher spoilage potential than A. allosaccharophila in sterile vacuum packed grouper fillets. • H. paralvei had a stronger ability to degrade proteins, utilize FAA, and produce VOCs. • Targeted inhibition of H. paralvei is beneficial to the preservation of grouper fillets. [ABSTRACT FROM AUTHOR]

As an emerging viral pathogen, Decapod iridescent virus 1 (DIV1) is a severe threat to the farmed and wild crustaceans, and it can spread between shrimp and crabs. Although swimming crab (Portunus trituberculatus) was identified as one of the DIV1 susceptible species by experimental infection mimicking the natural pathway, no case of natural infection was reported to date. Samples of Po. trituberculatus suffered from severe mortality were collected from a farm in Shandong Province of China. Diseased Po. trituberculatus exhibited slow movement and loss of appetite, while dying crabs showed empty stomachs and guts. Real-time PCR revealed that Po. trituberculatus samples were DIV1 positive and had a very high DIV1 load (up to 5.68 × 1010 copies/μg DNA) in gill tissue. Histopathological examination revealed eosinophilic inclusions and pyknosis in the hemocytes of the Po. trituberculatus gill tissue. Transmission electron microscopy of ultrathin sections showed that there were typical DIV1 particles in the gill tissue and hemocytes, which confirmed the disease of the farmed Po. trituberculatus was caused by the infection with Decapod iridescent virus 1 (iDIV1). Further bioassay by oral administration with tissues of diseased crabs and hemolymph coagulation examination showed that the diseased Po. trituberculatus had a significantly longer coagulation time than in healthy individuals. This study reported a severe case of natural infection with DIV1 in cultured swimming crab, further confirming the risk of DIV1 to the swimming crab farming industry and providing a new approach for on-site diagnosis of iDIV1 or other potential pathogens in Po. trituberculatus. • A disease outbreak of farmed Portunus trituberculatus has caused serious damage to this farm. • The evidence consistently confirmed that the farmed swimming crab were infected with DIV1. • Bioassay showed that the diseased Po. trituberculatus had a significantly longer coagulation time than in the healthy crab. • This study further confirming the risk of DIV1 to the swimming crab farming industry. [ABSTRACT FROM AUTHOR]

The immunomodulatory roles of Chinese herbal Medicine (CHM) in aquatic animals have been well-recorded. However, how CHM impacts the intestinal microbiota and serum metabolism is not fully understood. In this study, the effects of different additive levels of CHM on the growth performance, immunity, intestinal flora and serum metabolism of hybrid grouper (♀ Epinephelus fuscoguttatus × ♂ Epinephelus lanceolatu) were investigated. The addition of 0.5%, 1.0%, 1.5% and 2.0% Chinese herbal medicine compound to feed could significantly improve the weight gain rate (WGR), specific growth rate (SGR) and survival rate (SR) of grouper, reduced feed coefficient, while had no significant difference on morphometric parameter. The most significant improvement for the parameters above was observed in 1.5% group. Different addition levels of CHM could also significantly enhance the activities of ACP, AKP, SOD, CAT and LZM in serum. Accordingly, the supplementation of CHM significantly induced up-regulation of immune genes such as IL-8, IL-1β, TNF-α, Nrf2, Lzm in the liver, spleen and head kidney of grouper, improved the resistance of grouper to V. harveyi as well. The intestinal flora analysis showed that at the phylum level, the main dominant species of intestinal microorganisms were Firmicutes , Proteobacteria , Bacteroidota , Actinobacteriota , Gemmatimonadota , Desulfobacterota , Fusobacteriota and Myxococcota. At the genus level, the high abundance was Lactobacillus , Streptococcus , Bacteroides , Escherichia , Romboutsia , Sphingomonas and Muribaculaceae. The abundance of probiotics (such as Lachnospiraceae , Lactobacillaceae , Streptococcaceae , etc) in CHM-supplement groups were higher (highest in 1.5% group) compared with control group. Moreover, a total of 11 common differential metabolic pathways were screened by LC-MS metabolism analysis of serum, they were Neuroactive ligand-receptor interaction, Purine metabolism, Linoleic acid, Glycerophospholipid metabolism, Taurine and hypotaurine metabolism, Arginine and proline metabolism, ABC transporters, Aminoacyl-tRNA biosynthesis, Arachidonic acid metabolism, Drug metabolism-cytochrome P450, alpha-Linolenic acid metabolism. Also, three common differential metabolites (PI(20:4(5Z,8Z,11Z,14Z)/18:1(11Z)), PC(20:3(8Z,11Z,14Z)/22:1(13Z)), PC(22:0/20:4(5Z,8Z,11Z,14Z)) associated with intestinal health, growth and disease resistance was found. These data will contributes to a comprehensive understand for the regulatory roles of CHM on fish, which is also beneficial for the disease control and sustainable development of aquaculture. • Chinese herbal Medicine (CHM) supplement can improve the growth of hybrid grouper. • CHM supplement can enhance the expressions of immune genes, the activities of non-specific immune parameters and the resistance against bacterial infection of hybrid grouper. • CHM supplement can balance the intestinal flora and impact the serum metabolism profile of hybrid grouper. [ABSTRACT FROM AUTHOR]

Ranaviruses are nucleoplasmic large DNA viruses that can cause major economic losses in the aquaculture industry and pose a severe threat to global ecological diversity. The available literature demonstrates that classifiable members of the genus Ranavirus enter cells via multiple and complicated routes. Here, we demonstrated the underlying cellular entry mechanism of soft-shelled turtle iridovirus (STIV) using green fluorescence tagged recombinant virus. Treatment with chlorpromazine, sucrose, ethyl-isopropyl amiloride, chloroquine or bafilomycin A1 all significantly decreased STIV infection, suggesting that STIV uses clathrin-mediated endocytosis and macropinocytosis to enter cells via a pH-dependent pathway. Depletion of cellular cholesterol with methyl-β-cyclodextrin significantly inhibited STIV entry, but neither filipin III nor nystatin did, suggesting that STIV entry was cholesterol dependent but caveola independent. Treatment with dynasore, genistein, ML-7 or cytochalasin D all significantly inhibited STIV infection, indicating that Rac GTPase and myosin II activity were required for the macropinocytosis-like pathway as well as actin polymerization. Our findings suggest that the molecular events involved in STIV entry are not identical to those of other ranavirus isolates. Our results also extend our understanding of the molecular mechanism of iridovirus entry and pathogenesis. [ABSTRACT FROM AUTHOR]

A rapid risk assessment was conducted using a questionnaire composed of 10 questions asking experts in African swine fever (ASF) to identify and rank the potential risk factors associated with the introduction and spread of ASF in Japan. The experts participating in this risk assessment considered illegal food import, followed by transport routes and foreign workers, to be the most relevant pathway of ASF introduction into Japan. Kanto and Kyushu were identified as the most likely regions for ASF introduction. All experts agreed that China is the most likely source of ASF introduction into Japan. Most Japanese experts were of the view that the risk of ASF spread if introduced into Japan would be low, while foreign experts considered the risk to be moderate or high. Most experts answered that wild boars would play an important role in the persistence of ASF if the disease were to spread in Japan. [ABSTRACT FROM AUTHOR]

A new cell line (GS-1) was developed from the spleen tissue of the orangespotted grouper, Epinephelus coioides applied for viral infection studies of fish ranavirus and megalocytivirus. The cells proficiently multiplied in Leibovitz's L-15 medium supplemented with 10% fetal bovine serum at temperatures between 20°C and 32°C. Morphologically, the cell line comprised fibroblast-like cells, and this was confirmed by immunostaining with vimentin, fibronectin, and desmin antibodies. The optimal temperature for grouper iridovirus (GIV) and infectious spleen and kidney necrosis virus (ISKNV) proliferation in GS-1 cells was 25°C, and the highest titer of GIV was 108.4 TCID50/ml, and the highest titer of ISKNV was 105.2 TCID50/ml. Electron micrographs showed that the mean diameter of GIV virions was 180-220 nm, which was larger than ISKNV virions (160-200 nm). Negatively stained GIV particles possessed an envelope structure that was assembled by the three-layered structure with an inner electron-dense core surrounded by a lighter coat (mean diameter, 27 ± 3 nm). The highest GIV-induced mortality of groupers occurred at 25°C, whereas the highest ISKNV-induced mortality occurred at 30°C. In summary, GS-1 cell line is a valuable tool for isolating and investigating fish ranavirus and megalocytivirus in the same host system. [ABSTRACT FROM AUTHOR]

Abstract In April 2016, an outbreak emerged in a cultured population of black-spotted pond frog tadpoles in Shuangliu County, China, whereas tadpoles were suffering from substantial mortality (90%). Principal clinical signs of diseased tadpoles were comprised haemorrhage on their body surface, swollen abdomen with yellow ascites, congestion and swelling of the liver. The diseased tadpole's homogenates tissue were inoculated into epithelioma papulosum cyprini (EPC) cells at 25 °C for 4 days which caused typical cytopathic effect, and the viral titer TCID 50 reached 107/0.1 mL. In pathogenicity tests, tadpoles were immersed in 2‰ virus fluid for 8 h, the clinical signs were observed similar to those recognized in naturally infected tadpoles and mortality rate were reached up to 80%, which affirms that the virus was the main cause for this disease. In addition, transmission electron microscopy of EPC cells infected with isolated virus reflected that the virus was in a regular hexagon way (shape) with capsule like structure. The diagonal diameter was recorded 135 ± 8 nm, wherever virus particles were arrayed in crystalline manner in the cytoplasm. The electrophoresis of MCP gene PCR-product showed that the samples of diseased tadpoles, aquaculture water source and isolated virus were all positive. The sequence of the isolate revealed more than 99% similarities to ranavirus based on homology and genetic evolution analysis of the whole MCP gene, and the isolate belongs to FV3-like virus group. This study confirmed that ranavirus was the causative agent of this outbreak, and named the virus as Rana nigromaculata ranavirus (RNRV). Highlights • This is the first report of a ranaviral infection affecting Rana nigromaculata in China. • The isolated virus (Rana nigromaculata ranavirus, RNRV) can induce a high mortality in Rana nigromaculata by natural or artificial infection. • The RNRV belongs to FV3-like ranaviruses genogroup by MCP genes sequences analysis. [ABSTRACT FROM AUTHOR]

Andrias davidianus is a large and economically important amphibian in China. Ranavirus infection causes serious losses in A. davidianus farming industry. MicroRNA mediated host-pathogen interactions are important in antiviral defense. In this study, five small-RNA libraries from ranavirus infected and non-infected A. davidianus spleens were sequenced using high throughput sequencing. The miRNA expression pattern, potential functions, and target genes were investigated. In total, 1356 known and 431 novel miRNAs were discovered. GO and KEGG analysis revealed that certain miRNA target genes are associated with apoptotic, signal pathway, and immune response categories. Analysis identified 82 downregulated and 9 upregulated differentially expressed miRNAs, whose putative target genes are involved in pattern-recognition receptor signaling pathways and immune response. These findings suggested miRNAs play key roles in A. davidianus 's response to ranavirus and could provide a reference for further miRNA functional identification, leading to novel approaches to improve A. davidianus ranavirus resistance. [ABSTRACT FROM AUTHOR]

Zebrafish has become a popular research model in the last years, and several diseases affecting zebrafish research facilities have been reported. However, only one case of naturally occurring viral infections was described for this species. In 2015, infectious spleen and kidney necrosis virus (ISKNV) was detected in zebrafish from a research facility in Spain. Affected fish showed lethargy, loss of appetite, abnormal swimming, distention of the coelomic cavity and, in the most severe cases, respiratory distress, pale gills and petechial haemorrhages at the base of fins. Cytomegaly was the most relevant histopathological finding in organs and tissues, sometimes associated to degenerative and necrotic changes. ISKNV belongs to the relatively newly defined genus Megalocytivirus, family Iridoviridae, comprising large, icosahedral cytoplasmic DNA viruses. This is the first case of naturally occurring Megalocytivirus infection in zebrafish research facilities, associated with morbidity. The virus has been identified based on both pathologic and genetic evidence, to better understand the pathogenesis of the infection in zebrafish and the phylogenetic relationship with other iridoviruses. Given the ability of megalocytiviruses to cross-species boundaries, it seems necessary to implement stringent biosecurity practices as these infections may invalidate experimental data and have major impact on laboratory and cultured fish. [ABSTRACT FROM AUTHOR]

Abstract: A fundamental goal of disease ecology is to determine the landscape and environmental processes that drive disease dynamics at different biological levels to guide management and conservation. Although ranaviruses (family Iridoviridae) are emerging amphibian pathogens, few studies have conducted comprehensive field surveys to assess potential drivers of ranavirus disease dynamics. We examined the factors underlying patterns in site‐level ranavirus presence and individual‐level ranavirus infection in 76 ponds and 1,088 individuals representing five amphibian species within the East Bay region of California. Based on a competing‐model approach followed by variance partitioning, landscape and biotic variables explained the most variation in site‐level presence. However, biotic and individual‐level variables explained the most variation in individual‐level infection. Distance to nearest ranavirus‐infected pond (the landscape factor) was more important than biotic factors at the site level; however, biotic factors were most influential at the individual level. At the site level, the probability of ranavirus presence correlated negatively with distance to nearest ranavirus‐positive pond, suggesting that the movement of water or mobile taxa (e.g., adult amphibians, birds, reptiles) may facilitate the movement of ranavirus between ponds and across the landscape. Taxonomic richness associated positively with ranavirus presence at the site level, but vertebrate richness associated negatively with infection prevalence in the host population. This might reflect the contrasting influences of diversity on pathogen colonisation versus transmission among hosts. Amphibian host species differed in their likelihood of ranavirus infection: American bullfrogs (Rana catesbeiana) had the weakest association with infection while rough‐skinned newts (Taricha granulosa) had the strongest. After accounting for host species effects, hosts with greater snout–vent length had a lower probability of infection. Our study demonstrates the array of landscape, environmental, and individual‐level factors associated with ranavirus epidemiology. Moreover, our study helps illustrate that the importance of these factors varies with biological level. [ABSTRACT FROM AUTHOR]

Cationic antimicrobial proteins are an important part of innate nonspecific immunity. It is the first defensive level, which is inherent in almost all living organisms. The main objective of such proteins is the destruction of dangerous microorganisms (fungi, bacteria, viruses, parasites etc.). The skin of amphibians is a rich source of these molecules, which are produced and stockpiled in skin glands, which are usually located on the dorsal side of the body. Basically, they are spread over the surface of the body or grouped in special morphological structures – parotids. Currently the host defensive proteins were found in members of all families of amphibians, that suggests a connection among them with evolutionary advantages. Moreover, amphibian antimicrobial proteins can be used in modern medicine. Amphibians can become a rich source of biologically active agents and usage of them is very beneficial for pharmaceutical industry. These substances appeared to have much more abilities than it was believed before. For example, they can be used in methods of blood folding or antiviral therapy. Taking this into account, it is very promising to study antimicrobial proteins in Ukraine (from 15 anuran species of 5 families (Pelobatidae, Hylidae, Bufonidae, Ranidae and Bombinatoridae). This article describes the chemical structure and properties of the antimicrobial proteins presently known from the studies and their presence in different families of Anura. The main aim of the work is to show the variability of these substances in anurans to create a background for further investigations of amphibians' antimicrobial proteins in Ukraine and studying of their pharmaceutical potential. [ABSTRACT FROM AUTHOR]

A new cell line derived from the caudal fin of golden pompano Trachinotus ovatus (TOCF) was successfully established and characterized. TOCF cells grew well at 28° C in L‐15 medium supplemented with 10% foetal bovine serum (FBS). The cell line has been subcultured in more than 100 passages. Molecular characterization of 18S ribosomal (r)RNA and cytochrome oxidase subunit 1 (COI) confirmed that the TOCF cells were derived indeed from T. ovatus. TOCF cells have a modal chromosome number of 54. It was further showed that TOCF cells were transfected successfully with pEGFP‐N3 and pDsRED‐N1 plasmid, suggesting that TOCF cells could be used to research gene functions in vitro. Viral susceptibility tests showed that TOCF cells were susceptible to Singapore grouper iridovirus (SGIV), observed by the occurrence of the cytopathic effect (CPE) with the formation of inclusion bodies. In addition, the expression of major capsid protein (MCP) gene of SGIV changed during virus infection in TOCF cells. Thus, our present results described the characteristic of a TOCF cell line that could be a valuable tool for genetic manipulation, as well as isolation and propagation of iridovirus studies. [ABSTRACT FROM AUTHOR]

Predicting antigens that would be protective is crucial for the development of recombinant vaccine using genome based vaccine development, also known as reverse vaccinology. High-throughput antigen screening is effective for identifying vaccine target genes, particularly for pathogens for which minimal antigenicity data exist. Using red sea bream iridovirus (RSIV) as a research model, we developed enzyme - linked immune sorbent assay (ELISA) based RSIV-derived 72 recombinant antigen array to profile antiviral antibody responses in convalescent Japanese amberjack ( Seriola quinqueradiata ). Two and three genes for which the products were unrecognized and recognized, respectively, by antibodies in convalescent serum were selected for recombinant vaccine preparation, and the protective effect was examined in infection tests using Japanese amberjack and greater amberjack ( S. dumerili ). No protection was provided by vaccines prepared from gene products unrecognized by convalescent serum antibodies. By contrast, two vaccines prepared from gene products recognized by serum antibodies induced protective immunity in both fish species. These results indicate that ELISA array screening is effective for identifying antigens that induce protective immune responses. As this method does not require culturing of pathogens, it is also suitable for identifying protective antigens to un-culturable etiologic agents. [ABSTRACT FROM AUTHOR]

Shrimp hemocyte iridescent virus (SHIV) is a recently reported shrimp virus, which threats the cultured white-leg shrimp Litopenaeus vannamei and can cause huge economic loss in shrimp farming industry in China. A quantitative real time polymerase chain reactio (qPCR) assay was developed using a TaqMan probe to detect and quantify SHIV. A pair of qPCR primers, which amplify a 188 bp DNA fragment, and a TaqMan probe were selected from ATPase gene (ORF114R) of the SHIV genome. The primers and TaqMan probe used in this assay were shown to be specific for SHIV and did not react with White spot syndrome virus (WSSV), Infectious hypodermal and hematopoietic necrosis virus (IHHNV), Hepatopancreatic parvovirus (HPV), Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (VP AHPND ), and Enterocytozoon hepatopenaei (EHP), or healthy shrimp DNA. The detection limit of the qPCR method was as low as 4 copies per reaction. The diagnostic sensitivity and the diagnostic specificity of the qPCR compared with nested PCR were 95.3% and 99.2%, respectively. The resulting standard curves showed high correlation coefficient values (R 2  = 0.998) in the range of 4 × 10 9 to 4 × 10 0 DNA copies/reaction. Test of farm samples showed that SHIV was detected in L. vannamei , Fenneropenaeus chinensis and Macrobrachium rosenbergii contained SHIV ranged from 1.21E+02 to 7.95E+07 copies (μg DNA) −1 . Quantitative detection of different tissues in artificial infected shrimp showed that haemolymph contained the highest SHIV load and muscle contained lowest SHIV load. [ABSTRACT FROM AUTHOR]

The planktonic freshwater crustacean of the genus Daphnia are a model system for biomedical research and, in particular, invertebrate-parasite interactions. Up until now, no virus has been characterized for this system. Here we report the discovery of an iridovirus as the causative agent of White Fat Cell Disease (WFCD) in Daphnia. WFCD is a highly virulent disease of Daphnia that can easily be cultured under laboratory conditions. Although it has been studied from sites across Eurasia for more than 60 years, its causative agent had not been described, nor had an iridovirus been connected to WFCD before now. Here we find that an iridovirus--the Daphnia iridescent virus 1 (DIV-1)--is the causative agent of WFCD. DIV-1 has a genome sequence of about 288 kbp, with 39% G+C content and encodes 367 predicted open reading frames. DIV-1 clusters together with other invertebrate iridoviruses but has by far the largest genome among all sequenced iridoviruses. Comparative genomics reveal that DIV-1 has apparently recently lost a substantial number of unique genes but has also gained genes by horizontal gene transfer from its crustacean host. DIV-1 represents the first invertebrate iridovirus that encodes proteins to purportedly cap RNA, and it contains unique genes for a DnaJ-like protein, a membrane glycoprotein and protein of the immunoglobulin superfamily, which may mediate host-pathogen interactions and pathogenicity. Our findings end a 60-year search for the causative agent of WFCD and add to our knowledge of iridovirus genomics and invertebrate-virus interactions. [ABSTRACT FROM AUTHOR]

Red Sea bream iridovirus (RSIV) was initially isolated from marine fish, which belongs to Megalocytivirus , Iridoviridae . It can cause great economic losses in fish culture with high morbidity and mortality. In the present study, the pathogenicity and immune response associated with a RSIV genotype megalocytivirus infection were determined in mandarin fish ( Siniperca chuatsi ). Fish challenged showed typical clinical signs of iridovirus infection, including acute haemorrhages and enlarged visceral organs. Histopathological analysis revealed that extensive necrosis, vacuolization and inflammation were presented in the stomach, spleen, kidney and liver of the diseased fish. Blood cells counting and phagocytic assay indicated that the numbers of the red and white blood cells in the peripheral blood of infected fish increased significantly compared to the control group and the phagocytic percentage of leukocytes peaked at day 6 post infection. Quantitive real-time PCR (qRT-PCR) was also undertaken to analyse the host defensive response in mandarin fish challenged with RSIV. The expression level of ten genes including interferon-related factors (IRFs) IRF1 and IRF7 , Mx , Viperin , JAK1 , STAT1 , TCRα , TNFα , IL-1β and IL-8 during experimental infection were monitored at different point of time in liver, spleen and head kidney. Results revealed varying expression profiles and clear transcriptional activation of these immune related genes in different tissues, which will contribute to better understand the pathogenesis and host defensive system in iridovirus invasion. [ABSTRACT FROM AUTHOR]

As the major viral pathogen of grouper aquaculture, Singapore grouper iridovirus (SGIV) has caused great economic losses in China and Southeast Asia. In the previous study, we have generated highly specific ssDNA aptamers against SGIV-infected grouper spleen cells (GS) by Systematic Evolution of Ligands by Exponential Enrichment technology (SELEX), in which Q2 had the highest binding affinity of 16.43 nM. In this study, we would try to identify the specific sequences in the aptamer Q2 that exhibited the high binding affinity to SGIV-infected cells by truncating the original Q2 into some different specific segments. We first evaluated the specificity and binding affinity of these truncated aptamers to SGIV-infected cells by flow cytometry, fluorescent imaging of cells and aptamer-based enzyme-linked apta-sorbent assay (ELASA). We then performed cytotoxicity analysis, assessment of the inhibitory effects upon SGIV infection and the celluar internalization kinetics of each truncated aptamer. Compared to the initial Q2, one of the truncated aptamer Q2-C5 showed a 3-fold increase in the binding affinity for SGIV-infected cells, and held more effective inhibitory effects, higher internalization kinetics and stability. Hence, the aptamer’s truncated methods could be applied in the research of identifying aptamer’s key sequences. The shorter, structure optimizing aptamer showed more excellent performance over the originally selected aptamer, which could potentially be applied in developing commercial detection probes for the early and rapid diagnosis of SGIV infection, and highly specific therapeutic drugs against SGIV infection. [ABSTRACT FROM AUTHOR]

Rock bream iridovirus (RBIV) causes severe mass mortalities in rock bream ( Oplegnathus fasciatus ) in Korea. In this study, we investigated the potential of viral membrane protein to induce antiviral status protecting rock bream against RBIV infection. We found that fish administered with ORF008L (myristoylated membrane protein, MMP) vaccine exhibited significantly higher levels of survival compared to ORF007L (major capsid protein, MCP). Moreover, ORF008L-based DNA vaccinated fish showed significant protection at 4 and 8 weeks post vaccination (wpv) than non-vaccinated fish after infected with RBIV (6.7 × 10 5 ) at 23 °C, with relative percent survival (RPS) of 73.36% and 46.72%, respectively. All of the survivors from the first RBIV infection were strongly protected (100% RPS) from re-infected with RBIV (1.1 × 10 7 ) at 100 dpi. In addition, the MMP (ORF008L)-based DNA vaccine significantly induced the gene expression of TLR3 (14.2-fold), MyD88 (11.6-fold), Mx (84.7-fold), ISG15 (8.7-fold), PKR (25.6-fold), MHC class I (13.3-fold), Fas (6.7-fold), Fas ligand (6.7-fold), caspase9 (17.0-fold) and caspase3 (15.3-fold) at 7 days post vaccination in the muscle (vaccine injection site). Our results showed the induction of immune responses and suggest the possibility of developing preventive measures against RBIV using myristoylated membrane protein-based DNA vaccine. [ABSTRACT FROM AUTHOR]

The Jurassic Yanliao theropods have offered rare glimpses of the early paravian evolution and particularly of bird origins, but, with the exception of the bizarre scansoriopterygids, they have shown similar skeletal and integumentary morphologies. Here we report a distinctive new Yanliao theropod species bearing prominent lacrimal crests, bony ornaments previously known from more basal theropods. It shows longer arm and leg feathers than Anchiornis and tail feathers with asymmetrical vanes forming a tail surface area even larger than that in Archaeopteryx. Nanostructures, interpreted as melanosomes, are morphologically similar to organized, platelet-shaped organelles that produce bright iridescent colours in extant birds. The new species indicates the presence of bony ornaments, feather colour and flight-related features consistent with proposed rapid character evolution and significant diversity in signalling and locomotor strategies near bird origins. [ABSTRACT FROM AUTHOR]

Lymphocystis disease virus (LCDV) infection may induce a variety of host gene expression changes associated with disease development; however, our understanding of the molecular mechanisms underlying host-virus interactions is limited. In this study, RNA sequencing (RNA-seq) was employed to investigate differentially expressed genes (DEGs) in the gill of the flounder (Paralichthys olivaceus) at one week post LCDV infection. Transcriptome sequencing of the gill with and without LCDV infection was performed using the IlluminaHiSeq 2500 platform. In total, RNA-seq analysis generated 193,225,170 clean reads aligned with 106,293 unigenes. Among them, 1812 genes were up-regulated and 1626 genes were down-regulated after LCDV infection. The DEGs related to cellular process and metabolism occupied the dominant position involved in the LCDV infection. A further function analysis demonstrated that the genes related to inflammation, the ubiquitin-proteasome pathway, cell proliferation, apoptosis, tumor formation, and anti-viral defense showed a differential expression. Several DEGs including β actin, toll-like receptors, cytokine-related genes, antiviral related genes, and apoptosis related genes were involved in LCDV entry and immune response. In addition, RNA-seq data was validated by quantitative real-time PCR. For the first time, the comprehensive gene expression study provided valuable insights into the host-pathogen interaction between flounder and LCDV. [ABSTRACT FROM AUTHOR]

Poly (I:C) showed promise as an immunoprotective agents in rock bream against rock bream iridovirus (RBIV) infection. In this study, we evaluated the time-dependent virus replication pattern and antiviral immune responses in RBIV-infected rock bream with and without poly (I:C) administration. In the poly (I:C)+virus-injected group, virus copy numbers were more than 18.9-, 24.0- and 479.2-fold lower than in the virus only injected group at 4 (4.73 × 10 4 and 8.95 × 10 5 /μl, respectively), 7 (3.67 × 10 5 and 8.81 × 10 6 /μl, respectively) and 10 days post infection (dpi) (1.26 × 10 5 and 6.02 × 10 7 /μl, respectively). Moreover, significantly high expression levels of TLR3 (8.6- and 7.7-fold, at 4 and 7 dpi, respectively) and IL1β (3.6-fold at 2 dpi) were observed in the poly (I:C)+virus-injected group, but the expression levels were not significantly in the virus-injected group. However, IL8 and TNFα expression levels showed no statistical significance in both groups. Mx, ISG15 and PKR were significantly highly expressed from 4 to 10 dpi in the virus-injected group. Nevertheless, in the poly (I:C)+virus-injected group, Mx and ISG15 expression were significantly expressed from 2 dpi. In summary, poly (I:C) administration in rock bream induces TLR3, IL1β, Mx and ISG15-mediated immune responses, which could be a critical factor for inhibition of virus replication. [ABSTRACT FROM AUTHOR]

Due to high-density aquafarming in Taiwan, groupers are commonly infected with two different iridoviruses: Megalocytivirus (grouper iridovirus of Taiwan, TGIV) and Ranavirus (grouper iridovirus, GIV). Iridoviral diseases cause mass mortality, and surviving fish retain these pathogens, which can then be horizontally transferred. These viruses have therefore become a major challenge for grouper aquaculture. In this study, comparisons of the biological responses of groupers to infection with these two different iridoviruses were performed. A novel approach for transcriptomic analysis was proposed to enhance the discovery of differentially expressed genes and associated biological pathways. In this method, suitable and available reference species are selected from the NCBI taxonomy tree and the Ensembl and KEGG databases instead of either choosing only one model species or adopting the NCBI non-redundant dataset as references. Our results show that selection of multiple appropriate model species as references increases the efficiency and performance of analyses compared to those of traditional approaches. Using this method, 17 shared pathways and 5 specific pathways were found to be significantly differentially expressed following infection with the two iridoviruses, among which 11 pathways were additionally identified based on the proposed method of multiple reference species selection. Among the pathways responsive to infection with a specific iridovirus, the spliceosomal pathway (ko03040; p-value = 0.0011) was exclusively associated with TGIV infection, while the glycolysis/gluconeogenesis pathway (ko00010; p-value = 0.0032) was associated with GIV infection. These findings and designed corresponding biological experiments may facilitate a deeper understanding of the mechanisms by which both TGIV and GIV cause fatal infections, as well as the ways in which they induce different pathologies and symptoms. We believe that the proposed novel mechanism for de novo transcriptomic analysis provides superior and comprehensive functional annotations and that the resulting shared and specific pathways identified may help immunologists develop specific vaccines against various types of iridovirus in the near future. [ABSTRACT FROM AUTHOR]

Decapod iridescent virus 1 (DIV1) is a lethal virus that has a significant influence on the shrimp and prawn culture industries. The mechanism through which infected prawns respond to the DIV1 virus is currently unknown. Here, we examined in detail the clinical signs, histopathology, and humoral, cellular, and immune-related gene responses after a sub-lethal dose of DIV1 during the acute infection period of 0–120 h post infection (hpi). Interestingly, at the end of the experiment, DIV1-infected prawns had black lesions on several external regions. The DIV1-infected prawns also exhibited few karyopyknotic nuclei in the gills and intestine tissues and exhibited increasing immunological responses, as revealed by significant increases in all examined parameters, including total hemocytes, phagocytosis, lysozyme, and overall bactericidal activity, from 6 to 48 hpi. In addition, between 72 and 120 hpi, all immune response activities of DIV1-infected prawn were impaired compared with those of normal prawns, indicating negative impacts on immunological parameters. A viral load analysis of various tissues by qPCR indicated that hemocytes were the dominant initial viral target tissues, followed by the gills and hepatopancreas. An expression analysis of crucial immune-related genes by qRT‒PCR revealed various expression patterns in response to DIV1 infection; in particular, fold changes in the relative expression of anti-lipopolysaccharide factors (ALFs), prophenoloxidase (proPO), lipopolysaccharide and β-1,3-glucan binding protein (LGBP) were observed. Additionally, five common chemicals, calcium hypochlorite [Ca(OCl) 2 ] at 16.25–130 ppm, hydrogen peroxide (H 2 O 2) at 8.75–70 ppm, povidone iodine (PVP–I) at 3–24 ppm, benzalkonium chloride (BKC) at 20–160 ppm, and formalin at 25–200 ppm, had a significant effect on the killing of DIV1 particles in vitro within 24 h after exposure. These data will be helpful for determining the health status and immune defense mechanisms of giant river prawns during DIV1 infection periods. The study performed the first application of very common disinfectants, and the obtained information will be useful for implementing effective strategies to prevent and control DIV1 infection in both hatchery and grow-out ponds. • A sublethal dose of DIV1 isolated from wild black tiger shrimp was experimentally injected to giant river prawn. • A sublethal dose of DIV1 could not kill giant river prawn, but slightly affects to induce abnormal behaviors in a short period and external signs. • Induced giant river prawn can evidently induce immune parameters, immune-related gene expression, but slightly affects histopathological changes. • Five common disinfectants exhibit high efficacy in eliminating DIV1 within 24 h after exposure. [ABSTRACT FROM AUTHOR]

Rock bream iridovirus (RBIV) is a member of the Megalocytivirus genus that causes severe mortality to rock bream. Water temperature is known to affect the immune system and susceptibility of fish to RBIV infection. In this study, we evaluated the time dependent virus replication pattern and time required to completely eliminate virus from the rock bream body against RBIV infection at different water temperature conditions. The rock bream was exposed to the virus and held at 7 (group A1), 4 (group A2) and 2 days (group A3) at 23 °C before the water temperature was reduced to 17 °C. A total of 28% mortality was observed 24–35 days post infection (dpi) in only the 7 day exposure group at 23 °C. In all 23 °C exposure groups, virus replication peaked at 20 to 22 dpi (10 6 –10 7 /μl). In recovery stages (30–100 dpi), the virus copy number was gradually reduced, from 10 6 to 10 1 with faster decreases in the shorter exposure period group at 23 °C. When the water temperature was increased in surviving fish from 17 to 26 °C at 70 dpi, they did not show any mortality or signs of disease and had low virus copy numbers (below 10 2 /μl). Thus, fish need at least 50 days from peaked RBIV levels (approximately 20–25 dpi) to inhibit the virus. This indicates that maintaining the fish at low water temperature (17 °C) for 70 days is sufficient to eradicate RBIV from fish body. Thus, RBIV could be eliminated slowly from the fish body and the virus may be completely eliminated under the threshold of causing mortality. [ABSTRACT FROM AUTHOR]

Signal Transducer and Activator of Transcription 1 (STAT1) has been demonstrated to function as a critical mediator in multiple cell processes, such as cell proliferation, cell death, and innate immune response. Interestingly, two orthologues of human STAT1, including STAT1a and STAT1b genes have been identified in different fish. However, the detailed roles of fish STAT1a in virus replication still remained largely uncertain. Here, we cloned a STAT1a from orange-spotted grouper Epinephelus coioides (EcSTAT1a) and characterized its roles during fish virus infection. EcSTAT1a encoded a 751-aa peptide which shared 97% and 93% identity to STAT1 from mandarin fish ( Siniperca chuatsi ) and Malabar grouper ( Epinephelus malabaricus ), respectively. Amino acid alignment analysis showed that EcSTAT1a contained a STAT-int domain, a STAT-alpha domain, a STAT-bind domain (DNA binding domain), a SH2 domain and a STAT1-TAZ2 bind domain. In examined tissues from healthy grouper, the expression of EcSTAT1a was predominant in intestine, gill and liver. In grouper cells, the relative expression levels of EcSTAT1a was significantly increased during red-spotted grouper nervous necrosis virus (RGNNV) or Singapore grouper iridovirus (SGIV) infection. Under fluorescence microscopy, we found that EcSTAT1a mainly localized in the cytoplasm. The ectopic expression of EcSTAT1a in vitro significantly delayed the cytopathic effect (CPE) progression evoked by RGNNV and SGIV. Further studies showed that the expression levels of viral genes, including SGIV major capsid protein (MCP), VP19, ICP-18, LITAF and RGNNV coat protein (CP), RNA-dependent RNA polymerase (RdRp) were all significantly reduced in EcSTAT1a overexpressing cells compared to the control vector transfected cells, suggested that EcSTAT1a exerted antiviral activity against iridovirus and nodavirus. Furthermore, overexpression of EcSTAT1a significantly increased the expression of interferon related cytokines or effectors and pro-inflammatory factors. Together, our results elucidated that EcSTAT1a might function as a critical antiviral factor by regulating the host interferon immune and inflammation response. [ABSTRACT FROM AUTHOR]

Rock bream iridovirus (RBIV), which is a member of the Megalocytivirus genus, causes severe mass mortalities in rock bream in Korea. To date, the innate immune defense mechanisms of rock bream against RBIV is unclear. In this study, we assessed the expression levels of genes related to TLR9 and MyD88-dependent pathways in RBIV-infected rock bream in high, low or no mortality conditions. In the high mortality group (100% mortality at 15 days post infection (dpi)), high levels of TLR9 and MyD88 expressions (6.4- and 2.4-fold, respectively) were observed at 8 d and then reduced (0.6- and 0.1-fold, respectively) with heavy viral loads at 10 dpi (2.21 × 10 7 /μl). Moreover, TRAF6, IRF5, IL1β, IL8, IL12 and TNFα expression levels showed no statistical significance until 10 dpi. Conversely, in the low mortality group (28% expected mortality at 35 dpi), TLR9, MyD88 and TRAF6 expression levels were significantly higher than those in the control group at several sampling points until 30 dpi. Higher levels of IRF5, IL1β, IL8, IL12 and TNFα expression were also observed, however, these were not significantly different from those of the control group. In the no mortality group (0% mortality at 40 dpi), significantly higher levels of MyD88 (2 d, 4 d and 40 dpi), TRAF6 (2 dpi), IL1β (4 dpi) and IL8 (2 d and 4 dpi) expression were observed. In summary, RBIV-infected rock bream induces innate immune response, which could be a major contributing factor to effective fish control over viral transcription. MyD88, TRAF6, IL1β and IL8-related immune responses were activated in fish survivor condition (low or no mortality group). This is a critical factor for RBIV disease recovery; however, these immune responses did not efficiently respond in fish dead condition (high mortality group). [ABSTRACT FROM AUTHOR]

Previously, we observed that the transcripts of Invertebrate iridescent virus 6 (IIV6) are not polyadenylated, in line with the absence of canonical poly(A) motifs (AATAAA) downstream of the open reading frames (ORFs) in the genome. Here, we determined the 3′ ends of the transcripts of fifty-four IIV6 virion protein genes in infected Drosophila Schneider 2 (S2) cells. By using ligation-based amplification of cDNA ends (LACE) it was shown that the IIV6 mRNAs often ended with a CAUUA motif. In silico analysis showed that the 3′-untranslated regions of IIV6 genes have the ability to form hairpin structures (22–56 nt in length) and that for about half of all IIV6 genes these 3′ sequences contained complementary TAATG and CATTA motifs. We also show that a hairpin in the 3′ flanking region with conserved sequence motifs is a conserved feature in invertebrate-infecting iridoviruses (genus Iridovirus and Chloriridovirus). [ABSTRACT FROM AUTHOR]

Members of the family Iridoviridae, collectively referred to as iridovirids, are large, double-stranded DNA-containing viruses that infect invertebrates and cold-blooded (ectothermic) vertebrates. Infections in the former often lead to massive levels of virus replication resulting in iridescence of the infected animal and ultimately death. Among the latter, infections target a variety of organs and are capable of causing high levels of morbidity and mortality among commercially and ecologically important fish and amphibian species. The viral replication strategy has been elucidated primarily through the study of frog virus 3 (FV3) with additional input from other iridovirids of ecological or commercial importance. Replication occurs within both nuclear and cytoplasmic compartments and involves synthesis of genome length and concatemeric DNA, extensive methylation of the viral genome (among vertebrate viruses only), coordinate expression of three classes of viral gene products, and formation of icosahedral virions within cytoplasmic viral assembly sites. Phylogenetic analyses delineate five genera within the family and suggest that members of the families Iridoviridae, Ascoviridae, and Marseilleviridae compromise a monophyletic lineage in which ascoviruses are most closely related to invertebrate iridoviruses. [ABSTRACT FROM AUTHOR]

Virus encoded tumor necrosis factor receptor (TNFR) homologues are usually involved in immune evasion by regulating host immune response or cell death. Singapore grouper iridovirus (SGIV) is a novel ranavirus which causes great economic losses in aquaculture industry. Previous studies demonstrated that SGIV VP51, a TNFR-like protein regulated apoptotic process in VP51 overexpression cells. Here, we developed a VP51-deleted recombinant virus Δ51-SGIV by replacing VP51 with puro R -GFP. Deletion of VP51 resulted in the decrease of SGIV virulence, evidenced by the reduced replication in vitro and the decreased cumulative mortalities in Δ51-SGIV challenged grouper compared to WT-SGIV. Moreover, VP51 deletion significantly increased virus induced apoptosis, and reduced the expression of pro-inflammatory cytokines in vitro . In addition, the expression of several pro-inflammatory genes were decreased in Δ51-SGIV infected grouper compared to WT-SGIV. Thus, we speculate that SGIV VP51 functions as a critical virulence factor via regulating host cell apoptosis and inflammation response. [ABSTRACT FROM AUTHOR]

Ranaviruses are important pathogens of amphibians, reptiles and fish. To meet the need for an analytical method for generating normalised and comparable infection data for these diverse host species, two standard-curve based quantitative-PCR (qPCR) assays were developed enabling viral load estimation across these host groups. A viral qPCR targeting the major capsid protein (MCP) gene was developed which was specific to amphibian-associated ranaviruses with high analytical sensitivity (lower limit of detection: 4.23 plasmid standard copies per reaction) and high reproducibility across a wide dynamic range (coefficient of variation below 3.82% from 3 to 3 × 10 8 standard copies per reaction). The comparative sensitivity of the viral qPCR was 100% (n = 78) based on agreement with an established end-point PCR. Comparative specificity with the end-point PCR was also 100% (n = 94) using samples from sites with no history of ranavirus infection. To normalise viral quantities, a host qPCR was developed which targeted a single-copy, ultra-conserved non-coding element (UCNE) of vertebrates. Viral and host qPCRs were applied to track ranavirus growth in culture. The two assays offer a robust approach to viral load estimation and the host qPCR can be paired with assays targeting other pathogens to study infection burdens. [ABSTRACT FROM AUTHOR]

Portable user-friendly diagnostic tests can benefit detection and surveillance of wildlife diseases. Here, the performance of a compact POCKIT™ Nucleic Acid Analyzer for detection of Ambystoma tigrinum virus (ATV), an emerging Iridovirus that is associated with high host mortality in the western tiger salamander ( Ambystoma mavortium ) in North America was assessed. Tissue samples from 188 larval tiger salamanders collected from sites in Alberta, Canada were tested by both iiPCR and by conventional PCR. Results of the two assays showed 96.3% agreement. All 176 samples that tested positive by conventional PCR were also positive by iiPCR, while 12 of the samples that were negative by conventional PCR were positive by iiPCR. Comparison of the limits of detection of the two assays shows that the iiPCR assay was more sensitive than conventional PCR and had a LOD95 of 20 copies per reaction. The instrument automatically analyzes and displays results within 40 min following nucleic acid extraction. The novel technology could enhance detection of, and response to, wildlife pathogens, particularly those that occur sporadically, cause rapid outbreaks, and/or occur within isolated geographical regions. [ABSTRACT FROM AUTHOR]

DNA vaccination is one method to protect farmed fish from viral and bacterial diseases. Chimeric antigens encoded by DNA vaccines have been shown to increase the resistance to viral diseases. Here, we sequenced the gene encoding lysosome-associated membrane protein-1 from Japanese flounder, Paralichthys olivaceus , (JfLAMP-1) and assessed its use in a chimeric DNA vaccine fused with the major capsule protein (MCP) from red seabream iridovirus (RSIV). JfLAMP-1 cDNA has a length of 1248 bp encoding 415 aa, which contains transmembrane and cytoplasmic domains. JfLAMP-1 is constitutively expressed in several tissues and its expression in spleen was upregulated following injection of formalin-killed cells (FKC) of Edwardsiella tarda . Immunofluorescence analysis showed that JfLAMP-1 is distributed in the small and large granules in the cytoplasm and groups close to the nucleus. The DNA encoding the luminal domain of JfLAMP-1 was replaced with the gene for the RSIV MCP, and the construct was cloned in an expression vector (pCIneo). Fish vaccinated with pCLAMP-MCP had significantly higher antibody levels than fish vaccinated with pCIneo vector harboring the MCP gene (p < 0.05) at day 30 post-vaccination. [ABSTRACT FROM AUTHOR]

In this study, we evaluated the potential of poly (I:C) to induce antiviral status for protecting rock bream from RBIV infection. Rock bream injected with poly (I:C) at 2 days before infection (1.1 × 10 4 ) at 20 °C had significantly higher protection with RPS 13.4% and 33.4% at 100 and 200 μg/fish, respectively, through 100 days post infection (dpi). The addition of boost immunization with poly (I:C) at before/post infection at 20 °C clearly enhanced the level of protection showing 33.4% and 60.0% at 100 and 200 μg/fish, respectively. To investigate the development of a protective immune response, rock bream were re-infected with RBIV (1.1 × 10 7 ) at 200 dpi. While 100% of the previously unexposed fish died, 100% of the previously infected fish survived. Poly (I:C) induced TLR3 and Mx responses were observed at several sampling time points in the spleen, kidney and blood. Moreover, significantly high expression levels of IRF3 (2.9- and 3.1-fold at 1 d and 2 days post administration (dpa), respectively), ISG15 and PKR expression (5.4- and 10.2-fold at 2 dpa, respectively) were observed in the blood, but the expression levels were low in the spleen and kidney after poly (I:C) administration. Our results showed the induction of antiviral immune responses and indicate the possibility of developing long term preventive measures against RBIV using poly (I:C). [ABSTRACT FROM AUTHOR]

The Chinese giant salamander iridovirus (CGSIV), belonging to the genus Ranavirus in the family Iridoviridae, is the causative agent of an emerging infectious disease causing high mortality of more than 90% and economic losses in Chinese giant salamanders in China. In this study, a recombinant baculovirus-based vaccine expressing the CGSIV major capsid protein (MCP) was developed and its protective immunity in Chinese giant salamanders was evaluated. The recombinant Autographa californica nucleopolyhedrosis virus (AcNPV), expressing CGSIV MCP, designated as AcNPV-MCP, was generated with the highest titers of 1 × 108 plaque forming units/mL (PFU/mL) and confirmed by Western blot and indirect immunofluorescence (IIF) assays. Western blot analysis revealed that the expressed MCP reacted with mouse anti-MCP monoclonal antibodies at the band of about 53 kDa. The results of IIF indicated that the MCP was expressed in the infected Spodoptera frugiperda 9 (Sf9) cells with the recombinant baculovirus, and the Chinese giant salamander muscle cells also transduced with the AcNPV-MCP. Immunization with the recombinant baculovirus of AcNPV-MCP elicited robust specific humoral immune responses detected by ELISA and neutralization assays and potent cellular immune responses in Chinese giant salamanders. Importantly, the effective immunization conferred highly protective immunity for Chinese giant salamanders against CGSIV challenge and produced a relative percent of survival rate of 84%. Thus, the recombinant baculovirus expressing CGSIV MCP can induce significant immune responses involving both humoral and cell-mediated immunity in Chinese giant salamanders and might represent a potential baculovirus based vaccine candidate for Chinese giant salamanders against CGSIV. [ABSTRACT FROM AUTHOR]

Turbot aquaculture is a very important industry in China. However, it is hampered because of viral reddish body syndrome ( VRBS) and high mortality caused by piscine turbot reddish body iridovirus ( TRBIV). TRBIV virus is an icosahedron-like and cytoplasmic DNA virus, belonging to Iridoviridae, Megalocytivirus. In previous studies, we have identified two antigen mimotopes using bioinformatics and constructed prokaryotic expression vectors. In this study, a fragment of major capsid protein ( MCP) gene with the two antigenic epitopes was cloned into eukaryotic expression vector pVAX1, to generate a recombinant plasmid pVAX1- TRBIV- MCP. The plasmid DNA was transferred into turbot cell line TK using liposome, and transient expression was detected using RT- PCR. After injection into turbot ( Scophthalmus maximus), the expression of the antigen gene was analysed using RT- PCR and was shown to express in all tested tissues in vaccinated fish 2 and 7 days post-vaccination. The cumulative mortalities in the vaccinated and unvaccinated control fish were 30% and 88% respectively. Immune responses and upregulation of the expression of chemokine receptor, tumour necrosis factor, interferon and interferon-induced antiviral molecules were observed in the vaccinated fish 60 h post-vaccination. These results demonstrate that the vaccinated turbots had higher survival rate and produced specific serum antibodies following the TRBIV challenge. More studies are needed to develop and apply the promising DNA vaccine for virus control in turbot. [ABSTRACT FROM AUTHOR]

In this review we provide an overview of the diversity of large DNA viruses known to be pathogenic for invertebrates. We present their taxonomical classification and describe the evolutionary relationships among various groups of invertebrate-infecting viruses. We also indicate the relationships of the invertebrate viruses to viruses infecting mammals or other vertebrates. The shared characteristics of the viruses within the various families are described, including the structure of the virus particle, genome properties, and gene expression strategies. Finally, we explain the transmission and mode of infection of the most important viruses in these families and indicate, which orders of invertebrates are susceptible to these pathogens. [ABSTRACT FROM AUTHOR]

Rock bream iridovirus (RBIV) disease in rock bream ( Oplegnathus fasciatus ) remains an unsolved problem in Korea aquaculture farms. CpG ODNs are known as immunostimulant, can improve the innate immune system of fish providing resistance to diseases. In this study, we evaluated the potential of CpG ODNs to induce anti-viral status protecting rock bream from different RBIV infection conditions. We found that, when administered into rock bream, CpG ODN 1668 induces better antiviral immune responses compared to other 5 CpG ODNs (2216, 1826, 2133, 2395 and 1720). All CpG ODN 1668 administered fish (1/5 µg) at 2 days before infection (1.1 × 10 7 ) held at 26 °C died even though mortality was delayed from 8 days (1 µg) and 4 days (5 µg). Similarly, CpG ODN 1668 administered (5 µg) at 2 days before infection (1.2 × 10 6 ) held at 23/20 °C had 100% mortality; the mortality was delayed from 9 days (23 °C) and 11 days (20 °C). Moreover, when CpG ODN 1668 administered (1/5/10 µg) at 2/4/7 days before infection or virus concentration was decreased to 1.1 × 10 4 and held at 20 °C had mortality rates of 20/60/30% (2 days), 30/40/60% (4 days) and 60/60/20% (7 days), respectively, for the respective administration dose, through 100 dpi. To investigate the development of a protective immune response, survivors were re-infected with RBIV (1.1 × 10 7 ) at 100 and 400 dpi, respectively. While 100% of the previously unexposed fish died, 100% of the previously infected fish survived. The high survival rate of fish following re-challenge with RBIV indicates that protective immunity was established in the surviving rock bream. Our results showed the possibility of developing preventive measures against RBIV using CpG ODN 1668 by reducing RBIV replication speed (i.e. water temperature of 20 °C and infection dose of 1.1 × 10 4 ). [ABSTRACT FROM AUTHOR]

Wood frogs (Rana sylvatica) are highly susceptible to infection with Frog virus 3 (FV3, Ranavirus, Iridoviridae), a cause of mass mortality in wild populations. To elucidate the pathogenesis of FV3 infection in wood frogs, 40 wild-caught adults were acclimated to captivity, inoculated orally with a fatal dose of 104.43 pfu/frog, and euthanized at 0.25, 0.5, 1, 2, 4, 9, and 14 days postinfection (dpi). Mild lesions occurred sporadically in the skin (petechiae) and bone marrow (necrosis) during the first 2 dpi. Severe lesions occurred 1 to 2 weeks postinfection and consisted of necrosis of medullary and extramedullary hematopoietic tissue, lymphoid tissue in spleen and throughout the body, and epithelium of skin, mucosae, and renal tubules. Viral DNA was first detected (polymerase chain reaction) in liver at 4 dpi; by dpi 9 and 14, all viscera tested (liver, kidney, and spleen), skin, and feces were positive. Immunohistochemistry (IHC) first detected viral antigen in small areas devoid of histologic lesions in the oral mucosa, lung, and colon at 4 dpi; by 9 and 14 dpi, IHC labeling of viral antigen associated with necrosis was found in multiple tissues. Based on IHC staining intensity and lesion severity, the skin, oral, and gastrointestinal epithelium and renal tubular epithelium were important sites of viral replication and shedding, suggesting that direct contact (skin) and fecal-oral contamination are effective routes of transmission and that skin tissue, oral, and cloacal swabs may be appropriate antemortem diagnostic samples in late stages of disease (>1 week postinfection) but poor samples to detect infection in clinically healthy frogs. [ABSTRACT FROM AUTHOR]

North American amphibians have recently been impacted by two major emerging pathogens, the fungus Batrachochytrium dendrobatidis (Bd) and iridoviruses in the genus Ranavirus (Rv). Environmental factors and host genetics may play important roles in disease dynamics, but few studies incorporate both of these components into their analyses. Here, we investigated the role of environmental and genetic factors in driving Bd and Rv infection prevalence and severity in a biodiversity hot spot, the southeastern United States. We used quantitative PCR to characterize Bd and Rv dynamics in natural populations of three amphibian species: Notophthalmus perstriatus, Hyla squirella and Pseudacris ornata. We combined pathogen data, genetic diversity metrics generated from neutral markers, and environmental variables into general linear models to evaluate how these factors impact infectious disease dynamics. Occurrence, prevalence and intensity of Bd and Rv varied across species and populations, but only one species, Pseudacris ornata, harbored high Bd intensities in the majority of sampled populations. Genetic diversity and climate variables both predicted Bd prevalence, whereas climatic variables alone predicted infection intensity. We conclude that Bd is more abundant in the southeastern United States than previously thought and that genetic and environmental factors are both important for predicting amphibian pathogen dynamics. Incorporating both genetic and environmental information into conservation plans for amphibians is necessary for the development of more effective management strategies to mitigate the impact of emerging infectious diseases. [ABSTRACT FROM AUTHOR]

Although serine/threonine (ST) kinase is known to induce host cell death in GF-1 cells, it remains unclear how ST kinase induces mitochondrial function loss. In the present study, we addressed the issue of mitochondrial function loss by determining whether the Bcl-2 family members Bcl-2 and Bcl-xL can prevent ST kinase-induced cell death activity via interacting with the pro-apoptotic gene Bax. Grouper fin cells (GF-1) carrying EGFP-Bal-xL and EGFP-Bcl-2 fused genes were selected, established in cell culture, and used to examine the involvement of Bcl-2 and Bcl-xL overexpression in protection of GF-1 cells from the effects of the giant sea perch iridovirus (GSIV) ST kinase gene. Using the TUNEL assay, we found that EGFP-Bcl-2 and EGFP-Bcl-xL reduced GSIV ST kinase-induced apoptosis to 20% all at 24 h and 48 h post-transfection (pt). Also, Bcl-2 and Bcl-xL substantially reduced the percentage of cells with GSIV ST kinase-induced loss of mitochondrial membrane potential (Δψps) at 24 and 48 hpt, respectively, and this reduction correlated with a 30% and 50% enhancement of host cell viability at 24 and 48 hpt as compared with vector control. Moreover, analysis of the effect of Bcl-2 and Bcl-xL interaction with Bax targeted to mitochondria during ST kinase expression at 48 hpt found that Bcl-2 and Bcl-xL also interacted with Bax to block cytochrome c release. Finally, Bcl-2 and Bcl-xL overexpression caused blockage of ST kinase function at 48 hpt, which was correlated with preventing caspase-9 and -3 cleavage and activation, thereby blocking downstream death signaling events. Taken together, our results suggest that the ST kinase-induced Bax/mitochondria-mediated cell death pathway can be blocked by the interaction of Bcl-2 and Bcl-xL with Bax to inhibit cytochrome c release during MMP loss. This rescue activity also correlated with inhibition of caspase-9 and -3 activation, thereby enhancing cell viability. [ABSTRACT FROM AUTHOR]

Transmission is central to our understanding and efforts to control the spread of infectious diseases. Because transmission generally requires close contact, host movements and behaviors can shape transmission dynamics: random and complete mixing leads to the classic density-dependent model, but if hosts primarily interact locally (e.g., aggregate) or within groups, transmission may saturate. Manipulating host behavior may thus change both the rate and functional form of transmission. We used the ranavirus-wood frog ( Lithobates sylvaticus) tadpole system to test whether transmission rates reflect contacts, and whether the functional form of transmission can be influenced by the distribution of food in mesocosms (widely dispersed, promoting random movement and mixing vs. a central pile, promoting aggregations). Contact rates increased with density, as expected, but transmission rapidly saturated. Observed rates of transmission were not explained by observed contact rates or the density-dependent model, but instead transmission in both treatments followed models allowing for heterogeneities in the transmission process. We argue that contacts were not generally limiting, but instead that our results are better explained by heterogeneities in host susceptibility. Moreover, manipulating host behavior to manage the spread of infectious disease may prove difficult to implement. [ABSTRACT FROM AUTHOR]

Alien species are key vectors for the spread of globally emerging diseases, and these emerging diseases have proven to be devastating for amphibian populations world-wide. Border and post-border biosecurity activities are pivotal for preventing the introduction of new diseases, but their effectiveness has seldom been assessed., We developed and populated a model to describe transport pathways into Australia, and the biosecurity activities implemented to manage these pathways. We evaluated the capacity of Australian border and post-border biosecurity activities, frequently considered one of the best quarantine inspection services in the world, to prevent the introduction of alien ranaviruses via the unintentional transport of alien amphibians into six Australian States., High transport pressures, measured by the number of international airline passengers and ships arriving in each State, resulted in increasing total numbers of alien stowaway amphibians. Post-border detection of alien amphibians was variable across States and increased with the full-time equivalent employees devoted to post-border biosecurity in each State., The baseline probabilities of introduction, without biosecurity activities, for at least one infected amphibian into any State were very low (≤0·07 in all cases), January 2004-December 2012. The implementation of biosecurity activities reduced these introduction probabilities further, with reductions of up to 50% for some States., Synthesis and applications. We have demonstrated the efficacy of biosecurity activities in reducing the introduction risk of new diseases being transported unintentionally alongside alien amphibians. Critically, we found that not all alien amphibians had to be detected to reduce risks appreciably. We advocate the widespread adoption of border and post-border biosecurity activities to manage the risks posed by alien amphibians (and other stowaway species) as vectors of emergent diseases. We support the robust design of biosecurity activities by providing a framework to evaluate the likely outcomes of case-specific biosecurity arrangements. [ABSTRACT FROM AUTHOR]

Tripartite motif-containing 32 (TRIM32) has been demonstrated to pay vital roles in cancer, genetic disorders and antiviral immunity. However, the molecular functions of fish TRIM32 still remained largely unknown. Here, a novel TRIM32 gene from orange spotted grouper (EcTRIM32) was cloned and characterized. EcTRIM32 encoded a 685-aa protein which showed 93%, and 60% identity to large yellow croaker ( Larimichthys crocea ) and human ( Homo sapiens ), respectively. Amino acid alignment showed that EcTRIM32 contained a conserved RING-finger domain, a BBOX domain and NHL domain. In healthy grouper, the transcript of EcTRIM32 was predominantly detected in brain, liver, intestine, spleen and skin. After injection with Singapore grouper iridovirus (SGIV) and polyI:C, the relative expression of EcTRIM32 in grouper spleen was differently regulated, suggested that EcTRIM32 was involved in antiviral immune response. In transfected grouper spleen (GS) cells, EcTRIM32 displayed bright fluorescence aggregates or spots in the cytoplasm. Notably, the deletion RING domain altered its precise localization and distributed throughout the cytoplasm in GS cells. In EcTRIM32 overexpressing cells, the replication of SGIV or red-spotted grouper nervous necrosis virus (RGNNV) was significantly inhibited compared to the vector control cells. Moreover, the overexpression of EcTRIM32 positively regulated the interferon immune response, evidenced by the significant increase of the expression level of interferon related signaling molecules, including interferon regulatory factor 3 (IRF3), IRF7, interferon-stimulated gene 15 (ISG15), interferon-induced 35-kDa protein (IFP35), MXI, TIR-domain-containing adaptor-inducing interferon-β (TRIF) and melanoma differentiation-associated protein 5 (MDA5). Further studies showed that overexpression of EcTRIM32 significantly enhanced the MDA5-mediated interferon immune response, but decreased stimulator of interferon genes (STING)-mediated interferon immune response. Meanwhile, the expression levels of pro-inflammation cytokines, including TNFα, IL-6 and IL-8 were up-regulated by the ectopic expression of EcTRIM32. We speculated that the regulation of IRF7, and pro-inflammation cytokines by EcTRIM32 overexpression might contribute critical roles in SGIV infection. In addition, the deletion of RING domain not only significantly weakened the antiviral roles of EcTRIM32, but also obviously affected the regulatory effects of EcTRIM32 on interferon immune and inflammation response. Together, our results firstly demonstrated that fish TRIM32 acted as an antiviral factor against both DNA and RNA virus infection. [ABSTRACT FROM AUTHOR]

The European grapevine moth, Lobesia botrana (Denis & Schiffermüller) (Lepidoptera: Tortricidae) is one of the most harmful insect pests of grape species in many countries. The purpose of this study was to find a safe and effective microbial control agents against L. botrana. Pathogenicity of 4 bacterial, 12 fungal and 4 viral agents was tested on the 3rd instar larvae of L. botrana. Results showed that one strain of Bacillus thuringiensis (MnD) had the highest mortality (100%) on the larvae within 3 days of treatment. On the other hand, 93% mortality and mycosis with Metarhizium anisopliae (Gg-12) and 90% mortality with Chilo iridescent virus (CIV) were determined on the larvae of the pest. The findings indicate that B. thuringiensis (MnD), M. anisopliae (Gg-12) and C. iridescent virus (CIV) are considered suitable microbial control agents against L. botrana larvae. [ABSTRACT FROM AUTHOR]

Iridoviridae are known to cause disease in sturgeons in North America. Here, histological and molecular methods were used to screen for this family of virus in sturgeons from various European farms with low-to-high morbidity. Some histological samples revealed basophilic cells in the gill and labial epithelia, strongly suggesting the accumulation of iridovirus particles. Newly developed generic PCR tests targeting the major capsid protein ( MCP) gene of sturgeon iridoviruses identified in North America, namely the white sturgeon iridovirus and the Namao virus ( NV), produced positive signals in most samples from four sturgeon species: Russian ( Acipenser gueldenstaedtii), Siberian ( A. baerii), Adriatic ( A. naccarii) and beluga ( Huso huso). The sequences of the PCR products were generally highly similar one another, with nucleotide identities greater than 98%. They were also related to (74-88%), although distinct from, American sturgeon iridoviruses. These European viruses were thus considered variants of a single new virus, provisionally named Acipenser iridovirus-European (Ac IV-E). Moreover, three samples infected with Ac IV-E showed genetic heterogeneity, with the co-existence of two sequences differing by five nucleotides. One of our European samples carried a virus distinct from Ac IV-E, but closely related to NV identified in Canada (95%). This study demonstrates the presence of two distinct sturgeon iridoviruses in Europe: a new genotype Ac IV-E and an NV-related virus. [ABSTRACT FROM AUTHOR]

Streptococcus agalactiae causes systemic disease in a variety of wild and farmed fish, resulting in high levels of morbidity and mortality, as well as serious economic losses to the Nile tilapia aquaculture industry. The development of economic and applicable oral vaccines is therefore urgently needed for the sustainable development of Nile tilapia aquaculture. In this study, mesoporous silica nanoparticles (MSNs) were fabricated using sol–gel synthesis technology, and the antigens of surface immunogenic protein (Sip) was loaded into MSNs to develop a nanovaccine MSNs-Sip@HP55. The results showed that the prepared nanovaccine exhibited pH-controlled release, which could survive in the simulated gastric environment (pH 1.5), and release antigens in the simulated intestinal environment at pH 7.4. The nanovaccine could induce innate and adaptive immune responses in Nile tilapia. When the challenge doses were 1.5 × 106, 1.18 × 106, and 0.88 × 106 CFU/mL, the relative protection rates in immunized Nile tilapia were 63.33 %, 64.23 %, and 76.31 %, respectively. Taken together, the nanovaccine exhibited a high antigen utilization rate and was easily administered orally via feeding, which could protect Nile tilapia against challenge with S. agalactiae in large-scale farms. Oral vaccine based on MSNs carriers is a potentially promising strategy for the development of fish vaccines. • The vaccine exhibited a high antigen bioavailability based on the MSNs nano-carrier. • The nanovaccine delivery system had the advantages of pH-controlled release of antigens and good biocompatibility. • The nanovaccine, administered orally via feeding, could protect Nile tilapia against challenge with S. agalactiae. • The nanovaccine could stimulate innate and adaptive immunity. [ABSTRACT FROM AUTHOR]

The pathogen that caused the mass mortalities of Macrobrachium rosenbergii in many farms in Yangzhou, China from June to September 2021, was isolated from diseased prawns and identified as Decapod iridescent virus 1 (DIV1) by molecular diagnostics, phylogenetic analysis, transmission electron microscopy (TEM) observation, virulence test, and histopathological examination, which was named as DIV1-mr. The diseased prawns M. rosenbergii showed symptoms such as white spots under the carapace at the base of rostrum, redness of the hepatopancreas, and empty stomach and intestine. Histopathological observation revealed that the hepatopancreas, gills and intestines of the diseased prawns were severely damaged. Observation of TEM showed that the diseased prawns tissue contained a large number of icosahedral virus particles, which arranged regularly and neatly, and the virus were 175.5 ± 10.6 nm vertex to vertex, 166.6 ± 5.8 nm from face to face, with a nucleoid at 97.6 ± 2.4 nm. The amino acid sequence of the ATP ase of DIV1-mr is 100% homologous to that of Cherax quadricarinatus iridovirus (CQIV) and shrimp hemocyte iridescent virus (SHIV). The pathogenicity of DIV1-mr was determined by challenge experiments, and the median lethal dosage (LD50) of DIV1-mr for M. rosenbergii was calculated as 3.28 × 105 copies/mL. Viral metagenomics sequencing showed that the complete genome of DIV1-mr was 167,330 bp in length with 34.57% G + C content. It contains 181 predicted ORFs, which were associated with functional proteins. The study revealed that DIV1 was pathogen causing mass mortality of M. rosenbergii , which will contribute to the in-depth study of the epidemiological investigation, and pathogenic mechanism of DIV1 at the molecular level. • Mass mortalities of cultured Macrobrachium rosenbergii caused by Decapod iridescent virus 1 (DIV1) was reported. • The pathogenicity of DIV1-mr was determined by histopathological examination, LD50 and LT50. • The complete genome sequence of DIV1-mr was determined and analyzed for revealing its pathogenic mechanism at the molecular level. [ABSTRACT FROM AUTHOR]