Search

The shrimp farming sector has faced significant hurdles due to acute hepatopancreatic necrosis disease (AHPND). In this study, an attempt was made to combine the enzymatic recombinase amplification (ERA) assay and CRISPR-Cas12a (ERA-Cas12a) for the rapid detection of AHPND. A set of crRNA and ssDNA with high sensitivity and good specificity was screened, and the best optimized one-tube system was obtained. Our additional analysis focused on the sensitivity and specificity of ERA-Cas12a, contrasting it with the established methodology of the World Organization for Animal Health (WOAH). The ERA-Cas12a could deliver the desired outcomes in under 40 min with a detection limit of 2 × 103 copies/µL and showed no interaction with white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), Enterocytozoon hepatopenaei (EHP), Taura syndrome virus (TSV), covert mortality nodavirus (CMNV), and decapod iridescent virus 1 (DIV1). Our actual sample detection results showed that the ERA-Cas12a system has the advantage of being more intuitive and fast without losing too much sensitivity compared with the nested PCR and quantitative real-time PCR (qPCR). Moreover, the detection results could be directly observed under blue light irradiation. All these results indicate that ERA-Cas12a provides a rapid and specific diagnosis of AHPND, which could further reduce the dependence on the instrument and effectively reduce aerosol pollution. It could be helpful in the prevention of shrimp disease and in reducing the economic loss of shrimp aquaculture. [ABSTRACT FROM AUTHOR]

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

Pangolins have been found to carry severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2)‐related coronaviruses. In light of this discovery, interest has been piqued in viromes of these heavily trafficked wild animals. In this study, we performed viral metagenomic sequencing to explore viromes of both confiscated dead pangolins and captive healthy pangolins. Sequence reads of vertebrate‐associated viruses in Herpesviridae, Retroviridae, Iridoviridae, Reoviridae, Arenaviridae, and Flaviviridae were detected in confiscated dead pangolins. A novel rotavirus (RV) (Reoviridae), showing a high degree of genetic similarity to the RV species D (RVD) that was previously unreported in mammals, was further confirmed by using reverse transcription‐polymerase chain reaction (RT‐PCR) and Sanger sequencing. Three out of 18 samples from the confiscated dead pangolins were positive for genomic sequences of the novel RV. Importantly, sequence alignments and phylogenetic analyses demonstrated that these RV strains genetically belonged to the RVD. Nevertheless, these novel RVD strains were divergent from known RVD strains that have been found only in Avian. They formed a separate genetic cluster. Five serial passages were attempted to isolate the RV, but no live virus was obtained. In addition, fecal samples were collected from healthy pangolins (n = 41) in our institution and screened for RVs by viral metagenomic sequencing and RT‐PCR. In these fecal samples, neither species D nor previously identified species A RVs were detected. This study reported RVDs in pangolin samples for the first time to our knowledge. Identifiability disagreements between wild and captive pangolins highlight the need for further exploration into pangolin viruses to better understand their emergence and transmission potential. [ABSTRACT FROM AUTHOR]

As a newly discovered virus, Decapoda iridovirus 1 (DIV1) can cause a mortality rate of up to 100% in crustaceans, leading to huge economic losses. At present, there is no effective prevention and control measures for this disease. In the present study, the specific primers targeting highly conserved regions of MCP gene were designed, and then a quantitative real-time PCR method was established. The results indicate that DIV1 quantitative real-time PCR established has good specificity and does not cross react with other pathogens including white spot syndrome virus (WSSV), infectious subcutaneous and hematopoietic necrosis virus (IHHNV) and Vibrio parahaemolyticus induced acute hepatopancreatic necrosis disease (VpAHPND). The real-time PCR was capable of detecting DIV1 DNA at a minimum concentration of 10 copies/µL within 34 cycles. The method has good repeatability, with intra group and inter group coefficients of variation both less than 2%. Thirty-two clinical samples were assessed using both the real-time PCR and conventional PCR. The results shown real-time PCR we established are more sensitive than conventional PCR. In conclusion, this method has strong specificity, stable repeatability, and high sensitivity, providing technical support for clinical diagnosis, epidemiology investigation and monitoring of DIV1. [ABSTRACT FROM AUTHOR]

Simple Summary: Decapod iridescent virus 1 (DIV1), a new virus, has posed significant challenges to the Macrobrachium nipponense industry, and little is known about the mechanism of the host response to DIV1 infection. In order to understand the immune response of M. nipponense to DIV1 infection, transcriptome analysis was conducted on the hepatopancreas of M. nipponense to examine the global expression patterns at 48 hpi with DIV1. Our results showed that multiple immune-related genes (e.g., lectin, dorsal, wnt6, hsp70, integrin and caspase) may play a significant role in M. nipponense against DIV1 infection, and the immune-related signaling pathways were significantly activated. This study has the potential to enhance our comprehension of the immune response of M. nipponense to DIV1 infection, which is advantageous for future treatment strategies for disease caused by DIV1. Macrobrachium nipponense is a significant cultivated species in China. However, decapod iridescent virus 1 (DIV1), as a newly discovered crustacean-lethal virus, has resulted in significant financial losses for the M. nipponense industry. In order to examine the immunological response of M. nipponense to DIV1, we conducted transcriptome analysis of the hepatopancreas from M. nipponense infected with DIV1 using RNA-seq. RNA sequencing analysis identified a combined total of 41,712 assembled unigenes, and 7014 genes that showed differential expression were identified in the group infected with DIV1, compared to the control group. Among these DEGs, 3952 were found to be up-regulated, while 3062 were down-regulated; many well-characterized DEGs were involved in innate immune defense, particularly involving the C-type lectin receptor signaling pathway, complement and coagulation cascades, phagosome, lysosome and PPAR signaling pathway. Moreover, the expression levels of well-known immune-related genes (dorsal, wnt6, lectin, caspase, integrin, hsp70) in the hepatopancreas and hemolymph were investigated by Quantitative real-time PCR (qRT-PCR), and the findings demonstrated a significant increase in gene expression in the hepatopancreas and hemolymph at various time points after infection. The results acquired in this study offered further comprehensive understanding of the immunological response of M. nipponense to DIV1 infection. [ABSTRACT FROM AUTHOR]

Ranaviruses, members of the genus Ranavirus within the family Iridoviridae, have become a significant concern for amphibian populations globally, along with other cold‐blooded vertebrates, due to their emergence as a significant threat. We employed bioinformatics tools to examine the codon usage patterns in 61 DNA pol genes from Ranavirus, Lymphocystivirus, Megalocytivirus, and two unclassified ranaviruses, as no prior studies had been conducted on this topic. The results showed a slight or low level of codon usage bias (CUB) in the DNA pol genes of Ranavirus. Relative synonymous codon usage (RSCU) analysis indicated that the predominant codons favored in Ranavirus DNA pol genes terminate with C or G. Correlation analysis examining nucleotide content, third codon position, effective number of codons (ENC), correspondence analysis (COA), Aroma values, and GRAVY values indicated that the CUB across DNA pol genes could be influenced by both mutation pressure and natural selection. The neutrality plot indicated that natural selection is the primary factor driving codon usage. Furthermore, the analysis of the codon adaptation index (CAI) illustrated the robust adaptability of Ranavirus DNA pol genes to their hosts. Analysis of the relative codon deoptimization index (RCDI) suggested that Ranavirus DNA pol genes underwent greater selection pressure from their hosts. These findings will aid in comprehending the factors influencing the evolution and adaptation of Ranavirus to its hosts. [ABSTRACT FROM AUTHOR]

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

Staphylococcus aureus, a Gram-positive bacterium, causes inflammatory skin diseases, such as atopic dermatitis, and serious systemic diseases, such as sepsis. In the skin and nasal environment, peptidoglycan (PGN)-degrading enzymes, including lysozyme and lysostaphin, affects S. aureus PGN. However, the effects of PGN-degrading enzymes on the acute innate immune-inducing activity of S. aureus have not yet been investigated. In this study, we demonstrated that PGNdegrading enzymes induce acute silkworm hemolymph melanization by S. aureus. Insoluble fractions of S. aureus treated with lysozyme, lysostaphin, or both enzymes, were prepared. Melanization of the silkworm hemolymph caused by the injection of these insoluble fractions was higher than that of S. aureus without enzyme treatment. These results suggest that structural changes in S. aureus PGN caused by PGN-degrading enzymes affect the acute innate immune response in silkworms. [ABSTRACT FROM AUTHOR]

Large yellow croaker (Larimichthys crocea) is a vital marine‐cultured species in China. Large yellow croaker iridovirus (LYCIV) can cause a high mortality rate in L. crocea. Rapid and convenient detection of LYCIV is an urgent demand for diagnosis. In this study, rapid and simple recombinase polymerase amplification (RPA), real‐time RPA and RPA combined with lateral flow dipstick (RPA‐LFD) methods were developed for the detection of LYCIV based on the conserved sequence of the LYCIV major capsid protein (MCP) gene. With these optimized RPA analyses, LYCIV detection could be completed within 20 min at 40°C. Both RPA and real‐time RPA could detect viral DNA as low as 102 copies/μL, while the detection limit of RPA‐LFD was 101 copies/μL, and there was no cross‐reaction with other aquatic pathogens (KHV, CyHV‐2, GCRV‐JX01, SVCV, LCDV and LMBV). In practical evaluation of RPA, real‐time RPA and RPA‐LFD methods, the results showed consistency with the general PCR detection. In short, the developed RPA, real‐time RPA and RPA‐LFD analyses could be simple, rapid, sensitive and reliable methods for field diagnosis of LYCIV infection and have significant potential in the protection of LYCIV infection. [ABSTRACT FROM AUTHOR]

This article presents a new method for detecting Decapod iridescent virus 1 (DIV1), a harmful pathogen in crustacean farming. The method, called TaqMan-based real-time PCR, is highly accurate and specific for detecting aquatic viruses. The study developed primers and a probe targeting the DIV1 gene and tested the assay's specificity and sensitivity. The results showed that the method was highly specific and could detect very low levels of DIV1 DNA. The authors recommend using this method alongside another real-time PCR method for the best detection of DIV1. [Extracted from the article]

Simple Summary: Shrimp aquaculture is an important industry due to its advantages of short production cycles and good economic benefits, but it faces challenges from diseases caused by tiny organisms that can lead to slow growth or large-scale shrimp deaths. Three such harmful pathogens are Enterocytozoon hepatopenaei (EHP), infectious hypodermal and hematopoietic necrosis virus (IHHNV), and Decapod iridescent virus 1 (DIV1). It is essential to quickly identify these organisms. In this study, we created a new test that can detect all three pathogens at once by using a melting curve-based Eva Green real-time PCR assay. We used this test on 190 shrimp samples from different regions in China and found that a significant number of shrimp were affected by these organisms. Our test is as accurate as the current standard test but faster because it looks for all three organisms simultaneously. This new method can help shrimp farmers identify diseases early, which is crucial for treating sick shrimp and preventing the spread of disease. This work is valuable because it offers a better way to keep shrimp healthy, which benefits the shrimp industry and supports aquaculture biosecurity. Infections with Enterocytozoon hepatopenaei (EHP), infectious hypodermal and hematopoietic necrosis virus (IHHNV), and Decapod iridescent virus 1 (DIV1) pose significant challenges to the shrimp industry. Here, a melting curve-based triple real-time PCR assay based on the fluorescent dye Eva Green was established for the simultaneous detection of EHP, IHHNV, and DIV1. The assay showed high specificity, sensitivity, and reproducibility. A total of 190 clinical samples from Shandong, Jiangsu, Sichuan, Guangdong, and Hainan provinces in China were evaluated by the triple Eva Green real-time PCR assay. The positive rates of EHP, IHHNV, and DIV1 were 10.5%, 18.9%, and 44.2%, respectively. The samples were also evaluated by TaqMan qPCR assays for EHP, DIV1, and IHHNV, and the concordance rate was 100%. This illustrated that the newly developed triple Eva Green real-time PCR assay can provide an accurate method for the simultaneous detection of three shrimp pathogens. [ABSTRACT FROM AUTHOR]

Largemouth bass ranavirus (LMBRaV) belongs to the Ranavirus genus of the Iridoviridae family. It is a highly pathogenic virus that causes mass mortality in largemouth bass. In recent years, outbreaks of LMBRaV have been found in various provinces throughout China. Previous research mainly focused on virus isolation, identification, and detection, while the morphological change of the virus was still unknown. In this study, the ultrastructural morphogenesis of LMBRaV in epithelioma papulosum cyprinid (EPC) cells was observed and studied by using transmission electron microscopy. EPC cells were infected with LMBRaV (MOI=0.1) and then examined at 2 h, 4 h, 6 h, 12 h, 24 h, 48 h, 72 h and 96 h post infection. LMBRaV entered cells through endocytosis or direct penetration of cell membrane. After entering, the virus was observed in vesicles or lysosomes. After capsid uncoating, the virus genomes passed through the nuclear membrane and entered the cell nucleus. Virus genomes completed replication in the nucleus then transferred into the cytoplasm. In the cytoplasm, the progeny virus was assembled in the viromatrix and then aggregated in pseudocrystalline array. Finally, mature virus particles released through budding release from the cell membrane. Mature virus particles had a hexagonal shape and a diameter of approximately 150 nm. This study revealed the process of morphogenesis of LMBRaV in EPC cell line, providing essential information for further research on pathogenic mechanisms and immunological prevention of LMBRaV. [ABSTRACT FROM AUTHOR]

Introduction: Ranavirus disease, caused by viruses within the genus Ranavirus (Iridoviridae), is considered a globally emerging infectious disease linked to mass mortality events in both wild and cultured ectothermic vertebrates. Surveillance work is, however, limited in Asia hence prevalence and the dynamics of the disease remain poorly understood. To understand disease burden and the potential biotic and abiotic drivers in southern China region, we conducted a systematic surveillance of the ranavirus across Guangxi Zhuang Autonomous region (GAR). Methods: For this, we used a multifaceted approach involving screening of amphibians and other potential hosts, diagnostic tests, phylogenetic analyses, prevalence estimation, co-infection assessments, and climatic niche analyses. Over one thousand individuals were sampled across 25 sampling sites. Results: We found ninety-two individuals from 18 species of ectothermic vertebrates to be infected with ranavirus. Two lineages were responsible - Rana nigromaculata ranavirus and tiger frog virus were identified using phylogenetic analysis based on the major capsid protein (MCP) gene fragment. Out of these two lineages, the presence of tiger frog virus is rare as we came across only one case. We also found evidence of a co-infection with ranavirus and Batrachochytrium dendrobatidis that can be highly detrimental to host populations; possibly the first such documentation in Asia. Our niche modelling analysis suggests that precipitation seasonality plays an important role in ranavirus prevalence in GAR - southwestern, southeastern, central and northeastern regions of GAR can be considered to be optimum habitats for ranaviruses. Infection rates in wild frog species have reached 100% in some areas, even in nature reserves. Discussion: Our research also indicates that culture facilities and pet markets are frequently infected, serving as likely vectors for the regional and global spread of ranaviruses. The knowledge generated suggests the need for systematic surveillance, stringent biosecurity measures, and control of international animal trade to prevent further transmission and protection of biodiversity and aquaculture industries across Asia. [ABSTRACT FROM AUTHOR]

Chinese giant salamander iridovirus (GSIV) is the first known and causative viral pathogen in Andrias davidianus. Developing a sensitive, accurate and specific assay to detect GSIV in samples is essential to prevent the further spread of the pathogen. In this study, we established a droplet digital PCR (ddPCR) assay that targeted the mcp gene of GSIV, enabling rapid and quantitative detection of the virus. We determined that the optimal annealing temperature, primer concentration and probe concentration were 57.1°C, 50 nM and 500 nM, respectively. We analysed the specificity and sensitivity of the ddPCR assay and found that five common aquatic animal viruses, including Cyprinid herpesvirus 2 (CyHV‐2), infectious spleen and kidney necrosis virus (ISKNV), Koi herpesvirus (KHV) and Carp Edema Virus (CEV) displayed negative results based on this GSIV ddPCR assay. The assay can detect GSIV with the lowest detection limit of 3.7 copies per reaction. To evaluate the sensitivity and accuracy of the ddPCR assay, we tested different infected tissue samples with both the ddPCR and TaqMan real‐time PCR assays. Our results showed that the ddPCR assay detected GSIV in all samples with 100% positivity, while the TaqMan real‐time PCR assay detected GSIV in only 82.1% of samples. The established ddPCR method provided several advantages in detecting GISV, including high sensitivity, high precision and absolute quantification, making it a powerful tool for detection of possible and potential GSIV infection, even in samples with low viral load. [ABSTRACT FROM AUTHOR]

Stress-induced epidemic parasites and bacterial diseases are becoming a typical occurrence in fish aquaculture across the globe, those consider deadly infectious pathogens. To prevent losses on fish farms, it is crucial to obtain information on the infection severity and the characteristics of those diseases which have been peaked for fish farms and lead to mass mortality. This study investigates the effect of dual parasitic and bacterial infection on farmed tilapia, which leads to losses in farmed fish; focuses on the harmful effect of polluted water on speared infections in farming. During the production season, water and fish specimens were taken from private fish farms in El-Sharkia and Kafr El-Sheikh, Egypt, particularly during significant mortality times. The infected and uninfected fish samples were examined to determine bacteriological, parasitological, haemato-biochemical indices, immune responses, and pathologic changes besides water analysis in fish farming in the study area. It was discovered that fish were significantly infected with Centrocestus formosanus encysted metacercariae and/or Cichlidogyrus tilapiae, Heterophyes spp. Also, three species of gram-negative: Aeromonas hydrophila, A. sobria, Pseudomonas aeruginosa, and two Gram-positive cocci: Streptococcus agalactiae and Enterococcus faecalis. Identification of bacterial isolates was confirmed by using 16S rRNA gene sequencing. Results demonstrated substantial (P < 0.05) output losses in both farms. Results displayed negative effects of dual infection on the lost weight, whole-body biochemical, haemato-biochemical indices, digestive enzyme, immune responses, gene expression, and abnormal features histopathology in different organs of infected Nile tilapia. At the same time, most of the water Indices strayed considerably from standards. Thus, the obtained data indicated the detrimental impacts of dual infection and water quality deterioration on the health condition of Nile tilapia and might help us acquire a deeper understanding of yield loss as potentially severe economic repercussions for fish farms. [ABSTRACT FROM AUTHOR]

Objectives: This study was to identify and characterize Decapod iridescent virus 1 (DIV1) in the outbreaks reported in two whiteleg shrimp farms and one black tiger shrimp farm located in northern Taiwan in 2020. Methods: The histopathology, electron microscopy and polymerase chain reaction (PCR) specific for the DIV1 were used to identify the virus, and the phylogenetic analysis was performed by comparing the major capsid protein gene fragment of DIV1s from Taiwan with reference sequences of the family Iridoviridae. Results: DIV1 was identified by diagnostic PCR and caused mild mortality (20%) in cultured Penaeus monodon and high mortality (100%) in cultured whiteleg shrimp. Cultured P. monodon was first found to be infected with DIV1 through natural route of infection. Histopathological examination showed dark‐eosinophilic cytoplasmic inclusions in the degenerative cells of targeted hematopoietic tissues. For electron microscopy, a non‐enveloped virus particle was observed from homogenates of mixed target organs through negative staining with a diameter of 112±2 nm. Nucleotide sequences of DIV1 isolates from the Taiwanese outbreak are 100% identical to those from the PRC. Conclusions: Based on the clinical evidence, mortality rates, histopathology, electron microscopy examinations and phylogenetic analysis, it is believed that DIV1 is the causative agent of the outbreak. This is the first report of DIV1 in cultured shrimp in Taiwan. The emergence of DIV1 signals a warning to shrimp aquaculture farmers worldwide. [ABSTRACT FROM AUTHOR]

Decapod iridescent virus 1 (DIV1) is a dangerous new virus that causes mortality to shrimp and crayfish. This virus has been reported in China, which is a major exporter of decapods to South Korea and its neighboring countries, Taiwan and Thailand. This virus can be regarded as a pathogen with a high risk of entry into Korea. Therefore, South Korea has been conducting emergency quarantine for DIV1 since 2020, and in this study, an import risk analysis was performed following the World Organization of Animal Health and domestic laws and regulations to present scientific grounds and validity for South Korea's DIV1 emergency quarantine. Import risk analysis was performed by hazard identification, entry assessment, exposure assessment, and consequence assessment. As a result of the import risk analysis, it was confirmed that the current DIV1 quarantine can significantly reduce the import risk. However, it was also established that the management of prawns and polychaetes intended for animal feed or bait and currently not subjected to quarantine is also required. Additionally, two species of crabs that would meet the criteria for a completely susceptible species and two species of crayfish that required surveillance of the import amount or route of exposure were also identified. In conclusion, these uses and susceptible species were designated as quarantine targets for nonhuman consumption requiring risk management and import sanitary conditions to be performed before export and after import. Finally, alternatives to reduce the cost burden of stakeholders following the introduction of the system were also presented. [ABSTRACT FROM AUTHOR]

Invertebrate iridescent virus 6 (IIV6) is a member of the genus Iridovirus and belongs to the Iridoviridae family. The entirely sequenced dsDNA genome, composed of 212.482 bp, encodes 215 putative open reading frames (ORFs). ORF458R encodes a putative myristoylated membrane protein. RT-PCR analysis of ORF458R expression in the presence of DNA replication and protein synthesis inhibitors showed that this gene is transcribed in the late phase of the virus infection. Time course analysis showed that transcription of ORF458R initiates between 12 and 24 h p.i. and starts to decrease after this point. Transcription of ORF458R initiated 53 nucleotides upstream of the translation start site and ended 40 nucleotides after the stop codon. Dual luciferase reporter gene assay showed that sequences between ‒61st and +18th nucleotides are essential for promoter activity. Interestingly, a remarkable decrease in promoter activity, in the presence of sequences between ‒299th and ‒143rd nucleotides, suggested a repressor activity between these regions. Our results showed that ORF458R is transcriptionally active, and separately located sequences at its upstream region with promoter and repressor activities regulating its expression. This information on the transcriptional analysis of ORF458R will contribute to our understanding of the molecular mechanisms of IIV6 replication. [ABSTRACT FROM AUTHOR]

Aquaculture has expanded to become the fastest growing food-producing sector in the world. However, its expansion has come under threat due to an increase in diseases caused by pathogens such as iridoviruses commonly found in aquatic environments used for fish farming. Of the seven members belonging to the family Iridoviridae, the three genera causing diseases in fish comprise ranaviruses, lymphocystiviruses and megalocytiviruses. These three genera are serious impediments to the expansion of global aquaculture because of their tropism for a wide range of farmed-fish species in which they cause high mortality. As economic losses caused by these iridoviruses in aquaculture continue to rise, the urgent need for effective control strategies increases. As a consequence, these viruses have attracted a lot of research interest in recent years. The functional role of some of the genes that form the structure of iridoviruses has not been elucidated. There is a lack of information on the predisposing factors leading to iridovirus infections in fish, an absence of information on the risk factors leading to disease outbreaks, and a lack of data on the chemical and physical properties of iridoviruses needed for the implementation of biosecurity control measures. Thus, the synopsis put forth herein provides an update of knowledge gathered from studies carried out so far aimed at addressing the aforesaid informational gaps. In summary, this review provides an update on the etiology of different iridoviruses infecting finfish and epidemiological factors leading to the occurrence of disease outbreaks. In addition, the review provides an update on the cell lines developed for virus isolation and culture, the diagnostic tools used for virus detection and characterization, the current advances in vaccine development and the use of biosecurity in the control of iridoviruses in aquaculture. Overall, we envision that the information put forth in this review will contribute to developing effective control strategies against iridovirus infections in aquaculture. [ABSTRACT FROM AUTHOR]

Viral seasonality in the aquaculture industry is an important scientific issue for decades. While the molecular mechanisms underpinning the temperature-dependent pathogenesis of aquatic viral diseases remain largely unknown. Here we report that temperature-dependent activation of IL6-STAT3 signaling was exploited by grass carp reovirus (GCRV) to promote viral entry via increasing the expression of heat shock protein 90 (HSP90). Deploying GCRV infection as a model system, we discovered that GCRV induces the IL6-STAT3-HSP90 signaling activation to achieve temperature-dependent viral entry. Further biochemical and microscopic analyses revealed that the major capsid protein VP7 of GCRV interacted with HSP90 and relevant membrane-associated proteins to boost viral entry. Accordingly, exogenous expression of either IL6, HSP90, or VP7 in cells increased GCRV entry in a dose-dependent manner. Interestingly, other viruses (e.g., koi herpesvirus, Rhabdovirus carpio, Chinese giant salamander iridovirus) infecting ectothermic vertebrates have evolved a similar mechanism to promote their infection. This work delineates a molecular mechanism by which an aquatic viral pathogen exploits the host temperature-related immune response to promote its entry and replication, instructing us on new ways to develop targeted preventives and therapeutics for aquaculture viral diseases. Author summary: Viral seasonality in the aquaculture industry has been an important scientific issue for decades, which is causing billions of economic losses every year worldwide. However, there is a lack of understanding of how temperature influences the pathogenesis of aquatic viruses infecting fish, shellfish, and other ectotherms. Here, deploying aquatic grass carp reovirus (GCRV) as a model system, we demonstrate for the first time that the temperature-dependent IL6-STAT3-HSP90 signaling axis is exploited by GCRV to promote viral entry. We found that HSP90 interacted with viral structural proteins and membrane-associated proteins to enable viral entry. Furthermore, our study indicates other aquatic viruses might evolve a similar mechanism to promote their infection. By elucidating the molecular mechanism of temperature-dependent aquatic viral pathogenesis, our work may help to develop targeted prevention and control strategies against aquatic viral diseases. [ABSTRACT FROM AUTHOR]

Background: The house cricket, Acheta domesticus, is one of the most farmed insects worldwide and the foundation of an emerging industry using insects as a sustainable food source. Edible insects present a promising alternative for protein production amid a plethora of reports on climate change and biodiversity loss largely driven by agriculture. As with other crops, genetic resources are needed to improve crickets for food and other applications. Methods: We present the first high quality annotated genome assembly of A. domesticus from long read data and scaffolded to chromosome level, providing information needed for genetic manipulation. Results: Gene groups related to immunity were annotated and will be useful for improving value to insect farmers. Metagenome scaffolds in the A. domesticus assembly, including Invertebrate Iridescent Virus 6 (IIV6), were submitted as host-associated sequences. We demonstrate both CRISPR/Cas9-mediated knock-in and knock-out of A. domesticus and discuss implications for the food, pharmaceutical, and other industries. RNAi was demonstrated to disrupt the function of the vermilion eye-color gene producing a useful white-eye biomarker phenotype. Conclusions: We are utilizing these data to develop technologies for downstream commercial applications, including more nutritious and disease-resistant crickets, as well as lines producing valuable bioproducts, such as vaccines and antibiotics. [ABSTRACT FROM AUTHOR]

Mandarin fish ranavirus (MRV), as an emerging iridescent virus spread rapidly in China. To develop new anti-MRV strategies, in vitro and in vivo antiviral activities of lycorine were evaluated. In vitro, assays were conducted on mandarin fish brain-derived SCB3 cells. We found that lycorine exhibited therapeutic effect to prevent MRV replication, including the reduction of virus titters and suppression of MRV-MCP expression. Then, we sought to decipher the mechanisms underlying lycorine-mediated antiviral activities from the perspective of regulating apoptosis and type I interferon responses. JC-1 staining indicated that lycorine reversed the MRV-triggered collapse of MMP. Furthermore, lycorine defended the MRV-induced up-regulation of pro-apoptotic genes (Bad, Bax and p53) and down-regulation of anti-apoptotic genes (Bcl-2b, Bcl-xL and Mcl). As executor and initiator of apoptosis, caspase-3 and caspase-8 activities were inhibited by lycorine. Moreover, lycorine extinguished MRV-stimulated ROS to normal level. These founding argued that lycorine exerted the anti-MRV activities by regulating the mitochondrial apoptosis pathway. Subsequently, we confirmed that the expression of type I interferon pathway-related genes were activated by lycorine, which provided extra explanations to understand its anti-MRV mechanisms. In vivo assays, gavage administration of lycorine at doses above 19 mg/kg increased the survival rate of MRV-challenged mandarin fish from 20.00 % to 52.00 %, alongside with the reduction of MRV loads in spleen and head kidney. In addition, the activation of anti-apoptosis genes versus inhibition of pro-apoptosis genes was also found in the lycorine-treated fish. Overall, our study ascertained the activities of lycorine against MRV infection and provided new insights for discovering anti-MRV drugs. • Lycorine significantly resisted the MRV replication. • Lycorine exhibited prophylactic and therapeutic effects against in vitro MRV infection. • Lycorine activated the type I interferon pathway that was suppressed after MRV infection. • Lycorine inhibited MRV-induced mitochondrial apoptosis as well as ROS production. • Apoptosis may act as a potential target for discovering anti-MRV drugs. [ABSTRACT FROM AUTHOR]

Viral diseases are one of the important factors restricting the shrimp industry. Decapod iridescent virus 1 (DIV1) and White spot syndrome virus (WSSV) are the two most harmful viruses to shrimp. DIV1 and WSSV were found to be carried and infected simultaneously in several samples of cultured and wild shrimps. In this study, the co-infection model of DIV1 and WSSV in Litopenaeus vannamei was constructed by TaqMan probe quantitative detection (qPCR), intestinal section, and transcriptome analysis. Our study also evaluated the virulence of the virus by calculating LC 50 and detected the replication rates of DIV1 and WSSV at 7-time points and 2 tissues in L. vannamei by qPCR. As a result, DIV1 and WSSV could infect L. vannamei singly or co-infect L. vannamei. DIV1 replicated the fastest, at 12–16 h post-injection (hpi). The WSSV virus replicates the fastest 20–24 hpi. In different tissues, the amount of DIV1 carried by blood cells is higher than that in muscle, while WSSV is the opposite. In addition, the intestinal responses of DIV1 and WSSV single and co-infection of L. vannamei were explored using the RNA-Seq platform and bioinformatics analysis, and the differential expressed genes (DEGs) screened by comparison were as follows: there were significant differences in DEGs between the PBS group and the three virus infection groups, and there were differences in DEGs between the PBS group and single virus infection groups (the DIV1 group and the WSSV group), but there was no significant difference in the DEGs between the 2 single virus infection groups, and the Combine-infected group, especially, the DEGs between the WSSV group and the Combine-infected group were only 39. According to the above conclusions, it can be inferred that WSSV plays a major role in co-infection. In addition, pathways related to vitamin metabolism were significantly enriched in the viral infection group compared to the PBS group. Glycolysis/Gluconeogenesis, a hallmark of the Warburg effect, is also significantly enriched after viral infection. In this study, the co-infection phenomenon of DIV1 and WSSV was found for the first time in L. vannamei , which provided a new idea for developing and screening molecular markers for new strains resistant to complex viruses in L. vannamei. • The co-infection of DIV1 and WSSV in L. vannamei was reported for the first time. • The virulence of the virus was evaluated by drawing survival curve and calculating LC 50. • The fastest copy time period and organization of DIV1 and WSSV are revealed. • To reveal the mechanism of vitamin and Glycolysis/Gluconeogenesis pathway in co-infection. [ABSTRACT FROM AUTHOR]

The NFKBIE gene encodes the protein I-κBε, which is a key inhibitor of NF-κB in various immune organs and is crucial for the immune response to viruses. Grouper, a vital species in the marine economy, occupies a prominent position in the aquaculture industry. Nevertheless, the cultivation of grouper is currently encountering a substantial risk of disease, particularly from the neuronal necrosis virus (RGNNV) and Singapore grouper iridescent virus (SGIV). In this study, we investigated the molecular characterization of the EcNFKBIE gene in grouper, its function during RGNNV and SGIV infection, and the association between its SNP polymorphism and disease resistance. The molecular characterization results showed that EcNFKBIE exists a highly conserved structural domain AnK_2, exhibits ubiquitous cellular localization, and is expressed in all tissues of healthy grouper. Both in vitro and in vivo experiments have shown that the overexpression of EcNFKBIE effectively suppresses the expression of NF-κB family members (RELA, RELB, NF-κB1, NF-κB2, IKBα and c-Rel). And virus induced expression pattern analysis showed that EcNFKBIE expression is significantly activated during SGIV and RGNNV infection. Further overexpression and knockdown experiments indicated that EcNFKBIE promotes the replication of SGIV and RGNNVand aggravates the cytopathic effect. In addition, EcNFKBIE decreases the expression of interferon pathway genes and inflammatory factors, and suppresses the promoter activity of IFN3, ISRE and NF-κB. Generally, we suggest that EcNFKBIE is activated in response to RGNNV and SGIV, functioning to inhibit the NF-κB pathway and cytokine expression. This interference with cytokine activities allows the virus to evade the host's immune response, enhancing replication and achieving immune evasion. Furthermore, we explored the correlation between EcNFKBIE genomic DNA polymorphisms and resistance to RGNNV and SGIV diseases. Within the 12,428 bp nucleotide sequence, a total of 21 single nucleotide polymorphisms (SNPs) were identified, exhibiting predominantly moderate levels of polymorphism. Notably, one SNP was found to be linked to RGNNV resistance, while another was associated with SGIV resistance. These studies contribute to elucidating the mechanisms of fish virus immune evasion and to the development of scientific immunoprophylactic measures and breeding strategies for enhancing disease resistance in grouper. • We cloned and characterized the grouper EcNFKBIE, which is an inhibitor of NF-κB family members. • Overexpression of EcNFKBIE promotes SGIV and NNV replication in host immune cells. • EcNFKBIE helps viruses evade host immune response by inhibiting NF-κB pathway and cytokine expression. • EcNFKBIE's genome, at 12,428 bp, contains 21 SNPs, two linked to RGNNV and SGIV resistance. [ABSTRACT FROM AUTHOR]

The giant river prawn, Macrobrachium rosenbergii, is a major focus of aquaculture in tropical and sub‐tropical regions around the globe. Over the last 30 years, culture of M. rosenbergii has increased exponentially as demand has risen both for domestic consumption and for international export trade. As with many aquaculture species increases in production have been accompanied by the emergence of diseases affecting yield, profit and trading potential. Disease‐causing agents include pathogens infecting other crustaceans, such as Decapod Iridescent Virus (DIV1), as well as pathogens only known from M. rosenbergii such as White Tail Disease caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV). Here, we review the pathogenic agents associated with the culture of M. rosenbergii since commercial culture began in earnest during the 1970s. Particular emphasis is given to pathogens first identified in other aquaculture host species, but which have subsequently been shown to infect and cause disease in M. rosenbergii. As polyculture of M. rosenbergii with other aquaculture species is common practice, including culture with other decapods, crabs and fish, increased pathogen transfer among these farmed species may occur as M. rosenbergii aquaculture increases in the future. [ABSTRACT FROM AUTHOR]

Decapod iridescent virus 1 (DIV1), White spot syndrome virus (WSSV), and Enterocytozoon hepatopenaei (EHP) pose serious threats to the shrimp farming. To date, early detection remains an important way to control the occurrence and diffusion of these pathogens. Here, we developed for the first time, a loop‐mediated isothermal amplification (LAMP)‐based microfluidic chip detection system, which could detect DIV1, WSSV, and EHP simultaneously. The limits of detection (LoD) of the system were 10 copies/reaction for EHP and DIV1, and 102 copies/reaction for WSSV. The entire detection procedure could be completed rapidly in 40 min at 63°C with 100% specificity and had no cross‐reaction with other common shrimp pathogens. This newly established method was further validated using 94 Penaeus vannamei clinical samples, which were comparable to a typical qPCR assay and revealed good stability and reproducibility. These results illustrate that this LAMP microfluidic chip detection system allows rapid triplex pathogen analysis and could satisfy the demands of the field and routine diagnoses in aquaculture. [ABSTRACT FROM AUTHOR]

Largemouth bass (Micropterus salmoides) is an important commercial fish farmed in China. Challenges related to diseases caused by pathogens, such as iridovirus, have become increasingly serious. In 2017, we detected iridovirus-infected diseased largemouth bass in Zunyi, Guizhou Province. The isolated virus was identified as an infectious spleen and kidney necrosis virus (ISKNV)-like virus (ISKNV-ZY). ISKNV-ZY induces a cytopathic effect after infecting mandarin fish brain (MFB) cells. Abundant hexagonal virus particles were observed in the cytoplasm of ISKNV-ZY-infected MFB cells, using electron microscopy. The whole genome of ISKNV-ZY contained 112,248 bp and 122 open reading frames. Phylogenetic tree analysis showed that ISKNV-ZY was most closely related to BCIV, indicating that it is an ISKNV-like megalocytivirus. ISKNV-ZY-infected largemouth bass started to die on day six and reached a death peak on days 7–8. Cumulative mortality reached 100% on day 10. Using RNA sequencing-based transcriptome analysis after ISKNV-ZY infection, 6254 differentially expressed unigenes (DEGs) were identified, of which 3518 were upregulated and 2673 downregulated. The DEGs were associated with endocytosis, thermogenesis, oxidative phosphorylation, the JAK-STAT signaling pathway, the MAPK signaling pathway, etc. These results contribute to understanding the molecular regulation mechanism of ISKNV infection and provide a basis for ISKNV prevention. [ABSTRACT FROM AUTHOR]

At present, there are few studies on the epidemiology of diseases in wild Chinese white shrimp Penaeus chinensis. In order to enrich the epidemiological information of the World Organisation for Animal Health (WOAH)-listed and emerging diseases in wild P. chinensis, we collected a total of 37 wild P. chinensis from the Yellow Sea in the past three years and carried out molecular detection tests for eleven shrimp pathogens. The results showed that infectious hypodermal and hematopoietic necrosis virus (IHHNV), Decapod iridescent virus 1 (DIV1), yellow head virus genotype 8 (YHV-8), and oriental wenrivirus 1 (OWV1) could be detected in collected wild P. chinensis. Among them, the coexistence of IHHNV and DIV1 was confirmed using qPCR, PCR, and sequence analysis with pooled samples. The infection with YHV-8 and OWV1 in shrimp was studied using molecular diagnosis, phylogenetic analysis, and transmission electron microscopy. It is worth highlighting that this study revealed the high prevalence of coinfection with YHV-8 and OWV1 in wild P. chinensis populations and the transmission risk of these viruses between the wild and farmed P. chinensis populations. This study enriches the epidemiological information of WOAH-listed and emerging diseases in wild P. chinensis in the Yellow Sea and raises concerns about biosecurity issues related to wild shrimp resources. [ABSTRACT FROM AUTHOR]

Novel Decapod Iridescent virus (DIV1) infections emerged in mainland China around 2014 and have devastated shrimp aquaculture operations in Chinese coastal provinces. In 2020, DIV1 has spread to Taiwan with devastating results to shrimp and crayfish farms, in addition to being found in wild caught Penaeus monodon from the Indian Ocean. This trend is a major cause for concern and an urgent reminder to expand the tools needed to monitor the spread of DIV1 globally. Here, we describe a set of four different real‐time polymerase chain reaction (PCR) assays positioned across the genome of DIV1 to detect the virus in shrimp tissues. All four assays show a wide dynamic range and high analytical sensitivity and specificity. In addition, the newly developed assays show excellent diagnostic sensitivity and specificity in clinical Litopenaeus vannamei samples of North Asian origin. The new molecular toolset will enhance global capabilities to monitor the spread of DIV1 and ultimately be used as an early warning system for farmers and authorities to engage in appropriate risk mitigation strategies. [ABSTRACT FROM AUTHOR]

Introduction: Decapod iridescent virus 1 (DIV1) has caused severe economic losses in shrimp aquaculture. So far, Researchs on DIV1-infected shrimp have mainly focused on the hemocytes immune response, while studies on the host-intestine microbiota interactions during DIV1 infection have been scarce. Methods: This study determined the lethal concentration 50 (LC50) of DIV1 to Metapenaeus ensis, preliminarily determining that M. ensis could serve as a susceptible object for DIV1. The interactions and responses between the immune and intestine microbiota of shrimp under DIV1 infection were also investigated. Results and Discussion: DIV1 infection decreases intestine bacterial diversity and alters the composition of intestine microbiota. Specifically, DIV1 infection decreases the abundance of potentially beneficial bacteria (Bacteroidetes, Firmicutes, and Actinobacteria), and significantly increases the abundance of pathogenic bacteria such as Vibrio and Photobacterium, thereby increasing the risk of secondary bacterial infections. The results of PICRUSt functional prediction showed that altered intestine microbiota induces host metabolism disorders, which could be attributed to the bioenergetic and biosynthetic requirements for DIV1 replication in shrimp. The comparative transcriptomic analysis showed that some metabolic pathways related to host immunity were significantly activated following DIV1 infection, including ncRNA processing and metabolic process, Ascorbate and aldarate metabolism, and Arachidonic acid metabolism. M. ensis may against DIV1 infection by enhancing the expression of some immune-related genes, such as Wnt16, heat shock protein 90 (Hsp90) and C-type lectin 3 (Ctl3). Notably, correlation analysis of intestinal microbial variation with host immunity showed that expansion of pathogenic bacteria (Vibrio and Photobacterium) in DIV1 infection could increased the expression of NF-kB inhibitors cactus-like and Toll interacting protein (Tollip), which may limit the TLR-mediated immune response and ultimately lead to further DIV1 infection. Significance and Impact of the Study: This study enhances our understanding of the interactions between shrimp immunity and intestinal microbiota. The ultimate goal is to develop novel immune enhancers for shrimp and formulate a safe and effective DIV1 defense strategy. [ABSTRACT FROM AUTHOR]

A specimen of the non-indigenous species areolate grouper Epinephelus areolatus (Forsskål, 1775) was collected from the coastal waters off Latakia, Syria. This new finding of E. areolatus constitutes the second substantiated record of the species from the marine Syrian waters and the sixth for the Levant Basin and the entire Mediterranean Sea where a viable population appears to be successfully established at present. [ABSTRACT FROM AUTHOR]

Natural killer lysin (Nklysin) is a small molecule antimicrobial peptide produced by natural killer cells and T lymphocytes and widely expressed in vertebrates. Homologues of Nklysin have been found in several fish, but only several of biological activity was identified. In this study, we characterized a Nklysin from grouper (Epinephelus coioides), and explored its expression pattern and biological function in bacterial infection. We also investigated the role of Nklysin in viral replication and maturation. The nklysin gene of grouper encodes a 169 amino acid, sharing 92.90% identity to H. septemfasciatus NKlysin protein, containing a saposin B domain and six well-conserved cysteine residues that necessary for antimicrobial activity by forming three intrachain disulfide bonds. Analysis of qRT-PCR revealed that nklysin gene widely expressed in all tested tissues with the higher expressions in spleen. After bacterial challenge, the nklysin gene expression significantly varied in different tissues. In addition, a large-scale of the recombinant Nklysin protein was secreted in Pichia pastoris strain GS115. The MIC assay showed that the Nklysin protein directly inhibited growth of several pathogens, including Proteus mirabilis , Bacillus subtilis , Salmonella typhi , Escherichia coli , Shigella sonnei and Streptococcus agalactiae. Further analysis showed the Nklysin protein over-expression might prevent viral genes transcriptions and replication in FHM cells. Our findings suggested that the Nklysin of grouper might be a potential agent for antibacterial and antiviral infection in the future. • A homologue of nklysin gene was identified from grouper, Epinephelus coioides. • The nklysin gene could respond to bacterial challenge in immune tissue. • The Nklysin protein possessed a broad antibacterial spectrum. • Over-expression of Nklysin decreased virus replication in vitro. [ABSTRACT FROM AUTHOR]

Viral diseases have seriously restricted the healthy development of aquaculture, and decapod iridescent virus 1 (DIV1) has led to heavy losses in the global shrimp aquaculture industry. Due to the lack of effective treatment, early detection and regular monitoring are the most effective ways to avoid infection with DIV1. In this study, a novel real-time quantitative recombinase polymerase amplification (qRPA) assay and its instrument-free visualization improvement were described for the rapid detection of DIV1. Optimum primer pairs, suitable reaction temperatures, and probe concentrations of a DIV1-qRPA assay were screened to determine optimal reaction conditions. Then, its ability to detect DIV1 was evaluated and compared with real-time quantitative polymerase chain reactions (qPCRs). The sensitivity tests demonstrated that the limit of detection (LOD) of the DIV1-qRPA assay was 1.0 copies μL−1. Additionally, the presentation of the detection results was improved with SYBR Green I, and the LOD of the DIV1-RPA-SYBR Green I assay was 1.0 × 103 copies μL−1. Both the DIV1-qRPA and DIV1-RPA-SYBR Green I assays could be performed at 42 °C within 20 min and without cross-reactivity with the following: white spot syndrome virus (WSSV), Vibrio parahaemolyticus associated with acute hepatopancreatic necrosis disease (VpAHPND), Enterocytozoon hepatopenaei (EHP), and infectious hypodermal and hematopoietic necrosis virus (IHHNV). In conclusion, this approach yields rapid, straightforward, and simple DIV1 diagnoses, making it potentially valuable as a reliable tool for the detection and prevention of DIV1, especially where there is a paucity of laboratory equipment. [ABSTRACT FROM AUTHOR]

Groupers with an initial body weight of 9.10 ± 0.03 g were selected to investigate whether dietary addition of 0 (G0) and 1800 mg/kg glycerol monolaurate (GML, G1800) could alleviate the oxidative stress response and intestinal flora imbalance after 0, 6, 12, and 24 h of salinity change in grouper. Experimental results show that the dietary addition of GML significantly reduced the liver MDA content and increased the SOD activity of grouper. The gene expression of CAT and SOD increased and then decreased with time after adding 1800 mg/kg GML, and the highest values were significantly higher than those of the control group. Salinity change had a slight effect on the top four intestinal flora composition of grouper at 0, 12, and 24 h, with changes occurring only at 6 h when Cyanobacteria replaced Actinobacteria. The addition of dietary GML slowed down the intestinal flora disorder, inhibited the colonization of harmful bacterium Vibrio, and promoted the abundance of beneficial bacterium Bacillus. In conclusion, dietary GML significantly reduced the oxidative damage caused by sudden changes in salinity, improved the antioxidant capacity, and alleviated the intestinal flora imbalance in juvenile grouper. [ABSTRACT FROM AUTHOR]

Despite tens of cell lines originating from fish brain tissue have been constructed, little is known about the definite cell types they belong to. Whether fish cell lines derived from the brain shares similar characteristics is not well-answered yet. Here, we constructed three cell lines designated as LMB-S, LMB-M, LMB-L using brain tissue of spotted sea bass (Lateolabrax maculatus). Among them, LMB-L was identified as astroglia-like cells considering the high expression of GFAP, DCX, PTX, S100b, which are regarded as astrocyte-specific or astrocyte-associated cell markers. LMB-M exhibited smooth muscle-like features showing strong expression of LMOD1, SLAMP, M-cadherin, MGP, which are confirmed as muscle-restricted or myogenesis-involved cell markers. Although LMB-S was not definitely identified, it appeared an activation of WNT/β-catenin pathway. Besides the distinct expression profiles of cell markers, the three cell lines also presented differences in transfection efficiency and susceptibility to iridovirus infection. Relying on the established cell lines, a novel megalocytivirus, named LMIV (Lateolabrax maculatus iridovirus), was first isolated from diseased spotted sea bass. Genetic analysis of major capsid protein (MCP) and adenosine triphosphatase (ATPase) manifested that LMIV was clearly distinguishable from other representative teleost iridoviruses. Further investigations revealed that LMIV could replicate most efficiently in LMB-L cells obtaining the highest viral load (2.16 × 1010 copy/mL). By contrast, LMB-S cells gave rise to the highest viral load up to 3.86 × 108 copy/mL, when the three cell lines were infected with MRV, a newly emerged ranavirus. Moreover, LMIV infection caused lots of cells to be detached from monolayers, generating adherent and non-adherent cells. An opposite expression profiling of type I IFN pathway-related genes (JAK1, STAT1, STAT2, IRF9, Mx1) was found between adherent and non-adherent cells. Combined with the analysis of MCP gene expression, it is speculated that inhibiting type I IFN pathway in non-adherent cells allowed the facilitation of virus duplication. Taken together, the present study broadens our understanding about the diversity of cell lines derived from fish brain tissue and screening cells more susceptible to virus is not only meaningful for the development of vaccine, but also provide clues for further clarification of cell-iridovirus interactions. • Three cell lines from the brain of spotted sea bass were developed. • Two of cell lines were identified as astroglia-like and smooth muscle-like cells. • A novel megalocytivirus designated as LMIV was first isolated. • Inhibiting Type I IFN pathway probably facilitated LMIV duplication. [ABSTRACT FROM AUTHOR]

European North Atlantic ranavirus (ENARV, Iridoviridae), is a ranavirus species recently isolated from lumpfish (Cyclopterus lumpus, L.), which are used as cleaner fish in Atlantic salmon (Salmo salar) farming in Northern Europe. This study aimed to investigate (1) the virulence of ENARV isolates from Ireland, Iceland and the Faroe Islands to lumpfish; (2) horizontal transmission between lumpfish; and (3) virulence to Atlantic salmon parr. Lumpfish were challenged in a cohabitation model using intraperitoneally (IP) injected shedders, and naïve cohabitants. IP challenge with isolates from Iceland (1.9 × 107 TCID50 ml−1) and the Faroe Islands (5.9 × 107 TCID50 ml−1) reduced survival in lumpfish, associated with consistent pathological changes. IP challenge with the Irish strain (8.6 × 105 TCID50 ml−1) did not significantly reduce survival in lumpfish, but the lower challenge titre complicated interpretation. Horizontal transmission occurred in all strains tested, but no clinical impact was demonstrated in cohabitants. Salmon parr were challenged by IP injection with the Irish isolate, no virulence or virus replication were demonstrated. A ranavirus qPCR assay, previously validated for fish ranaviruses, was first used to detect ENARV in tissues of both in lumpfish and Atlantic salmon. This study provides the first data on the assessment of virulence of ENARV isolates to lumpfish and salmon, guidelines for the diagnosis of ENARV infection, and poses a basis for further investigations into virulence markers. [ABSTRACT FROM AUTHOR]

In a context where the search for plant-derived additives is a hot topic, glycerol monolaurate (GML) was chosen as our subject to study its effect on grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂). Seven gradient levels of GML (0, 600, 1200, 1800, 2400, 3000, and 3600 mg/kg) were used for the experiment. Based on our experiments, 1800 mg/kg GML significantly increased the final body weight (FBW) and weight gain rate (WGR). GML increased the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and decreased malondialdehyde (MDA). Adding 1800 mg/kg GML also significantly increased the levels of lauric acid (C12:0) (LA), n-3 polyunsaturated fatty acids (PFA), and the n-6 PFA-to-n-3/n-6 ratio, while significantly decreasing the levels of saturated fatty acids (SFA). Dietary supplementation with GML significantly inhibited the expression of pro-inflammatory factors and reduced the occurrence of inflammation. GML improved intestinal flora and the abundance of beneficial bacteria (Bacillus, Psychrobacter, Acinetobacter, Acinetobacter, Stenotrophomonas, and Glutamicibacter). It provides a theoretical basis for the application of GML in aquafeed and greatly enhances the possibility of using GML in aquafeed. Based on the above experimental results, the optimum level of GML in grouper feed is 1800 mg/kg. [ABSTRACT FROM AUTHOR]

White spot disease (WSD) has posed a serious threat to the China and the global shrimp aquaculture. In order to diagnose white spot syndrome virus (WSSV) early and prevent the spread and outbreak of WSD, it is necessary to establish a highly sensitive WSSV diagnosis method suitable for shrimp farming sites. In this study, a pre-amplification qPCR assay from the crude extract of samples heated lysis was established, which was further compared with the universal qPCR assay to verify the shrimp samples. The limit of detection (LOD) of pre-amplification qPCR assay and universal qPCR assay was 2.80 copies and 20.57 copies per reaction at 95% CI, respectively. It had good WSSV specificity and did not show cross-detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV), hepatopancreatic parvovirus (HPV), Enterocytozoon hepatopenaei (EHP), acute hepatopancreas necrosis disease (AHPND), necrotizing hepatopancreatitis bacteria (NHPB), and decapod iridescent virus 1 (DIV1). A total of 36 shrimp samples were detected as WSSV DNA positive by pre-amplification qPCR with crude extract from samples heated lysis and universal qPCR with DNA extraction. The diagnostic sensitivity and specificity were 97.22% (85.5 ~ 99.9%, 95% CI) and 100% (81.5 ~ 100%, 95% CI), respectively. The agreement Kappa value was 0.959 (0.879 ~ 1, 95% CI), and the analysis results were basically consistent. Eliminating the tedious steps of extracting DNA and using pre-amplified qPCR to detect WSSV in shrimp, it is a good choice for aquaculture farms. [ABSTRACT FROM AUTHOR]

Macrobrachium rosenbergii , the largest freshwater palaemonid prawn globally, suffers from mass mortalities in aquaculture production due to Decapod iridescent virus 1 (DIV1) infection in recent year. It is known that water temperature is a crucial factor affecting DIV1 replication. Nevertheless, current knowledge regarding the influence of temperature on DIV1 replication in different tissues of M. rosenbergii and the host immune response remains limited. In this study, we assessed the DIV1 copies in different sensitive tissues of M. rosenbergii under various temperatures, which revealed that the optimal temperature for DIV1 proliferation was 25 °C and 28 °C, and there was no proliferation at 34 °C. In different sensitive tissues, the viral load increased most remarkably between 24 and 48 h post-infection (hpi), and reached the peak at 72 hpi. Hemocytes were the most sensitive tissue, followed by hepatopancreas, gills and intestines, with maximum viral loads of 3.15 × 109, 1.41 × 109, 3.32 × 108 and 1.23 × 108 copies/ng at 72 hpi, respectively. Furthermore, the immune response of M. rosenbergii infected by DIV1 was analyzed, and significant immune-related pathways and genes involved in antiviral defense processes were evaluated based on the transcriptome analysis of the hepatopancreas. The results showed that 141 and 952 genes were significantly up-regulated, and 190 and 1343 genes were significantly down-regulated at 48 and 72 hpi, respectively, indicating a more intricate and robust immune response at 72 than 48 hpi. KEGG pathway enrichment analysis showed that some well-recognized immune-related pathways, such as Lysosome pathway and Phagosome pathway, were highly activated, revealing a complex immune response. Meanwhile, DIV1 infection may also lead to metabolic disorders. In addition, six immune-related genes (HSP70 , PI3K , SOD , Lectin , Lamp1 and Caspase) were selected for further determination, and the analysis revealed that these highly significant up-regulated genes played crucial antiviral roles. The results of this study revealed the effects of water temperature on viral replication dynamics and the immune responses of M. rosenbergii at the molecular level. • The influence of water temperature on viral replication dynamics of DIV1 in tissues of M. rosenbergii was studied. • The immune response of infected M. rosenbergii was reported. • The expression of six immune-related genes (HSP70 , PI3K , SOD , Lectin , Lamp1 and Caspase) was determined by qPCR, and their crucial antiviral roles were analyzed. [ABSTRACT FROM AUTHOR]

[Display omitted] • The newly developed nested PCR method eliminates the risk of false positive results inherent in the original methods. • The assay is highly specific and the detection limit was down to 1.37 × 101 copies/reaction. • The assay has DSe and DSp of 100 % and 99.7 %, respectively, in comparison to the reported real-time PCR. • The assay meets the Stage II evaluation of WOAH and is more suitable for inclusion in international diagnostic standards. DIV1 has the characteristics of fast transmission and a broad host range. Its infection leads to a high mortality rate, posing a serious threat to the global crustacean aquaculture industry. In order to increase the accuracy of DIV1 detection and reduce the difficulty of result interpretation, this study modified the original nested PCR method targeting the DIV1 ATPase gene. The internal primers for the nested PCR were redesigned to produce a 338 bp amplification product in the second step PCR, effectively distinguishing the target band from primer dimers. The newly established nested PCR method exhibits strong specificity and high sensitivity, with a detection limit as low as 1.37 × 101 copies/reaction. The developed nested PCR assay provides new technical support for the accurate detection of DIV1 in global crustacean aquaculture. [ABSTRACT FROM AUTHOR]

In recent years, with global warming and increasing marine pollution, some novel marine viruses have become widespread in the aquaculture industry, causing huge losses to the aquaculture industry. Decapod iridescent virus 1 (DIV1) is one of the newly discovered marine viruses that has been reported to be detected in a variety of farmed crustacean and wild populations. Several previous studies have found that DIV1 can induce Warburg effect-related gene expression. In this study, the effects of DIV1 infection on intestinal health of shrimp were further explored from the aspects of histological, enzymatic activities, microorganisms and metabolites using Marsupenaeus japonicus as the object of study. The results showed that obvious injury in the intestinal mucosa was observed after DIV1 infection, the oxidative and antioxidant capacity of the shrimp intestine was unbalanced, the activity of lysozyme was decreased, and the activities of digestive enzymes were disordered, and secondary bacterial infection was caused. Furthermore, the increased abundance of harmful bacteria, such as Photobacterium and Vibrio, may synergized with DIV1 to promote the Warburg effect and induce metabolic reprogramming, thereby providing material and energy for DIV1 replication. This study is the first to report the changes of intestinal microbiota and metabolites of M. japonicus under DIV1 infection, demonstrating that DIV1 can induce secondary bacterial infection and metabolic reprogramming. Several bacteria and metabolites highly associated with DIV1 infection were screened, which may be leveraged for diagnosis of pathogenic infections or incorporated as exogenous metabolites to enhance immune response. [ABSTRACT FROM AUTHOR]

Decapod iridescent virus 1 (DIV1) is an emerging viral pathogen that seriously threatens crustacean farming and has caused enormous economic losses in recent years. This virus was originally isolated and identified from diseased crayfish Cherax quadricarinatus and shrimp Litopenaeus vannamei in Fujian and Zhejiang, China, in 2014 by two research groups. In 2019, the Executive Committee of the International Committee on Taxonomy of Viruses approved DIV1 as a member of the new genus Decapodiridovirus in the family Iridoviridae. DIV1 is icosahedral in shape and contains a single linear double‐stranded DNA genome of approximately 165 kb. DIV1 can infect many host species, including freshwater and seawater crustaceans, but infected hosts do not show unique pre‐clinical signs, which increases the difficulties in the diagnosis and treatment of this disease. Increasing progress has been made towards understanding many aspects of this virus, including its classification, genomic features, transmission mode, host range, pathogenicity, clinical symptoms, histopathology, detection method and host immunity in response to infection. In this review, we provide an overview of recent progress towards understanding the epidemiology and pathobiology of disease caused by DIV1 and highlight the future research prospects in control and prevention strategies. [ABSTRACT FROM AUTHOR]

A member of the Iridoviridae family has been detected as the erythrocytic necrosis virus (ENV), which causes viral erythrocytic necrosis (VEN) in 20 marine and anadromous fishes. The major capsid protein (MCP) is the main structural protein of iridoviruses and is responsible for causing disease in various fishes. It has been found that the VEN utilizes major capsid protein (MCP) to enter the host cell and blocking the virus entry by targeting the protein can reduce the economic losses caused by the pathogen. The main objective of the study was to evaluate the inhibitory potentiality of 48 compounds Allium sativum is one of the medicinal plants which has been reported to show potential antiviral activity against various pathogens, but activity against the capsid protein promoted pathogens has not yet been reported. The MCP was retrieved, modeled, refined, and validated in this experiment. The binding affinity of 48 compounds was calculated against the MCP with the docking, ADMET, and molecular dynamics (MD) simulation approaches which predict PubChem CID 12303662 inhibitory compound that binds strongly with the major capsid protein with a binding affinity of -9.7 after optimization. For theoretical calculation, the HOMO-LUMO gap score was also calculated. The best ADMET compounds were selected for the optimization analysis and re-docking. As a result of the research, it is possible to deduce that these Allium sativum phytochemicals might act as significant inhibitors of the MCP. More in-vitro testing is needed to establish their effectiveness. [ABSTRACT FROM AUTHOR]

As nucleocytoplasmic large DNA viruses, replication of ranaviruses (genus Ranavirus, family Iridoviridae) involves a series of viral and host proteins. We have described that the replication and transcription machinery of Andrias davidianus ranavirus (ADRV) which was isolated from the Chinese giant salamander contained host factors. Here, a new host factor, the MutS homolog 2 (MSH2), was proved as an important protein that participated in ADRV infection. Expression of MSH2 was stable during ADRV infection in cultured cells and it localized at the cytoplasmic viral factories and colocalized with virus nascent DNA, indicating its possible role in virus genome replication. Investigation of the viral proteins that interacted with MSH2 by co-immunoprecipitation showed that A. davidianus MSH2 can interact with ADRV-35L (possible components associated with virus transcription), ADRV-47L (virus DNA polymerase), and ADRV-98R. Further knockdown MSH2 expression by RNAi significantly reduced the late gene expression of ADRV. Additionally, MSH2 knockout by CRISPR/Cas9 significantly reduced viral titers, genome replication, and late gene transcription of ADRV. Thus, the current study proved that ADRV can engage cellular MSH2 for its efficient genome replication and late gene transcription, which provided new information for understanding the roles of host factors in ranavirus replication and transcription. [ABSTRACT FROM AUTHOR]

The Andrias davidianus ranavirus (ADRV) is a member of the family Iridoviridae and belongs to the nucleocytoplasmic large DNA viruses. Based on genomic analysis, an ADRV-encoding protein, ADRV 12L, and its homologs from other iridoviruses were predicted as Rad2 family proteins based on the conserved amino acids, domains, and secondary structures. Expression analysis showed that the transcription of ADRV 12L started at 4 h post infection, and its expression was not inhibited by a DNA-replication inhibitor. Meanwhile, immunofluorescence localization showed that ADRV 12L mainly localized in viral factories and colocalized with the viral nascent DNA, which hinted at a possible role in DNA replication. Furthermore, a mutant ADRV lacking 12L (ADRV-Δ12L) was constructed. In both luciferase assays based on homologous recombination (HR) and double-strand break repair (DSBR) that followed, ADRV-Δ12L induced less luciferase activity than the wild-type ADRV, indicating that HR and DSBR were impaired in ADRV-Δ12L infected cells. In addition, infection with ADRV-Δ12L resulted in smaller plaque sizes and lower viral titers than that with wild-type ADRV, indicating an important role for 12L in efficient virus infection. Therefore, the results suggest that Rad2 homologs encoded by iridovirus have important roles in HR- and DSBR-process of the viral DNA and, thus, affect virus replication and the production of progeny virions. [ABSTRACT FROM AUTHOR]

Iridoviruses are DNA virus and have caused huge economic losses in the aquaculture industry. The aim of this study was to establish a colorimetric loop-mediated isothermal amplification (LAMP) protocol for the on-site detection of Singapore grouper iridovirus (SGIV). The SGIV-VP61 gene was chosen as the target gene to develop a colorimetric LAMP assay. The optimized condition of the colorimetric LAMP assay was incubation at 63?C for 1 h. Samples infected with SGIV could be detected with the color change from yellow into pink. The sensitivity of the developed assay is 5.66 copies/µL of the viral DNA template. This sensitivity was about 1000 times higher than that of conventional PCR while it was slightly lower than the one-step semi-nested PCR assay. A total of 60 DNA samples extracted from the fin tissue of the SGIV-infected Asian seabass were examined for SGIV by colorimetric LAMP, semi-nested PCR and conventional PCR. The results of the colorimetric LAMP assay showed 94.87 % agreement with the semi-nested PCR. In addition, the DNA extraction method using NaOH showed a better performance in the colorimetric LAMP assay. Taken together, the colorimetric LAMP established was a sensitive, rapid and specific method for the detection of SGIV. SGIV was not detected in samples randomly taken from a genetically improved line of the Asian seabass. However, some seabass obtained from the local markets were found to contain SGIV. Thus, the LAMP assay has the potential application in the diagnosis of iridovirus diseases in the aquaculture industry. [ABSTRACT FROM AUTHOR]

[Display omitted] • Two shrimp proteins, i.e. cuticle protein 8-like (CP8) and glutamine-fructose-6-phosphate aminotransferase [isomerizing] 2-like isoform X7, were identified to interact with DIV1-168L. • The expression of CP8 was mainly distributed in gill and epidermis. • The expression level of CP8 was significantly inhibited during DIV1 infection. Decapod iridescent virus 1 (DIV1) stands as a significant pathogen affecting crustaceans, posing a grave threat to the shrimp industries in aquaculture dependent nations. Within the Iridoviridae family, the conserved envelope protein DIV1-168L plays a pivotal role in virion entry. Nonetheless, the host factors that interact with 168L remain unidentified. To address this gap, we established a cDNA library derived from Litopenaeus vannamei gill tissue and conducted yeast two-hybrid screening to identify host factors that interact with 168L. Additionally, we performed co-immunoprecipitation assays to verify the interaction between cuticle protein 8 (CP8) and 168L. Expression pattern analysis revealed the presence of CP8 transcripts in the gill and epidermis. Furthermore, immunohistochemistry results demonstrated the expression of CP8 in gill cells and its localization in the gill filament epithelium. Fluorescence analysis indicated that full-length CP8 colocalized with 168L in the cytoplasm of Sf9 cells. Removal of the signal peptide from the N -terminal of CP8 eliminated its concentration in the cytoplasm. Additionally, CP8 expression was significantly inhibited during DIV1 infection. Therefore, our research contributes to a better understanding of the entry mechanism of iridovirids. The GenBank accession number for the DIV1 sequence is MF197913.1. [ABSTRACT FROM AUTHOR]