Search Results (20)

Red sea bream iridovirus is a serious threat to farmed fish, but little is known about how repeated freezing and thawing affect its stability. This study investigated the effects of repeated freeze–thaw cycles on RSIV infectivity by comparing quantitative polymerase chain reaction (qPCR), viability qPCR (vqPCR), and 50% tissue culture infectious dose (TCID₅₀) assays. While qPCR detected high amounts of viral DNA after multiple cycles, both viability qPCR and TCID₅₀ revealed a significant loss of infectivity unless serum was present. Correlation analysis showed a high degree of agreement between vqPCR and TCID₅₀, indicating their high compatibility for assessing viral infectivity. However, the correlations between qPCR and vqPCR, as well as between qPCR and TCID₅₀, were significantly lower, suggesting that qPCR alone may overestimate viral infectivity by detecting non-infectious viral DNA. These results demonstrate the critical role of serum in preserving RSIV infectivity and highlight the superior accuracy of vqPCR and TCID₅₀ in assessing viral infectivity compared with qPCR. This study emphasizes the importance of serum in storage media and suggests that combining vqPCR with TCID₅₀ is a more reliable measure of RSIV infectivity than qPCR alone. Full article

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

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

Long non-coding RNAs (lncRNAs) play crucial roles in a variety of biological processes, including stress response. However, the number, characteristics and stress-related expression of lncRNAs in turbot are still largely unknown. In this study, a total of 12,999 lncRNAs were identified at the genome-wide level of turbot for the first time using 24 RNA-seq datasets. Sequence characteristic analyses of transcripts showed that lncRNA transcripts were shorter in average length, lower in average GC content and in average expression level as compared to the coding genes. Expression pattern analyses of lncRNAs in 12 distinct tissues showed that lncRNAs, especially lincRNA, exhibited stronger tissue-specific expression than coding genes. Moreover, 612, 1351, 1060, 875, 420 and 1689 differentially expressed (DE) lncRNAs under Vibrio anguillarum, Enteromyxum scophthalmi, and Megalocytivirus infection and heat, oxygen, and salinity stress conditions were identified, respectively. Among them, 151 and 62 lncRNAs showed differential expression under various abiotic and biotic stresses, respectively, and 11 lncRNAs differentially expressed under both abiotic and biotic stresses were selected as comprehensive stress-responsive lncRNA candidates. Furthermore, expression pattern analysis and qPCR validation both verified the comprehensive stress-responsive functions of these 11 lncRNAs. In addition, 497 significantly co-expressed target genes (correlation coefficient (R) > 0.7 and q-value < 0.05) for these 11 comprehensive stress-responsive lncRNA candidates were identified. Finally, GO and KEGG enrichment analyses indicated that these target genes were enriched mainly in molecular function, such as cytokine activity and active transmembrane transporter activity, in biological processes, such as response to stimulus and immune response, and in pathways, such as protein families: signaling and cellular processes, transporters and metabolism. These findings not only provide valuable reference resources for further research on the molecular basis and function of lncRNAs in turbot but also help to accelerate the progress of molecularly selective breeding of stress-resistant turbot strains or varieties. Full article

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. Full article

Red sea bream iridovirus (RSIV) causes significant economic losses in aquaculture. Here, we analyzed the pathogenicity, viral shedding, and transmission dynamics of RSIV in rock bream (Oplegnathus fasciatus) by employing immersion infection and cohabitation challenge models. Rock bream challenged by immersion exposure exhibited 100% mortality within 35 days post RSIV exposure, indicating that the viral shedding in seawater peaked after mortality. At 25 °C, a positive correlation between the viral loads within infected rock bream and virus shedding into the seawater was observed. Specific RSIV lesions were observed in the spleen and kidney of the infected rock bream, and the viral load in the spleen had the highest correlation with the histopathological grade. A cohabitation challenge mimicking the natural transmission conditions was performed to assess the virus transmission and determine the pathogenicity and viral load. The RSIV-infected rock breams (donors) were cohabited with uninfected rock bream, red sea bream (Pagrus major), and flathead grey mullet (Mugil cephalus) (recipients) at both 25 °C and 15 °C. In the cohabitation challenge group maintained at 15 °C, no mortality was observed across all experimental groups. However, RSIV was detected in both seawater and the recipient fish. Our results provide preliminary data for further epidemiological analyses and aid in the development of preventive measures and management of RSIVD in aquaculture. Full article

Heat shock proteins 70 (HSP70s) are known to play essential roles in organisms’ response mechanisms to various environmental stresses. However, no systematic identification and functional analysis has been conducted for HSP70s in the turbot (Scophthalmus maximus), a commercially important worldwide flatfish. Herein, 16 HSP70 genes unevenly distributed on nine chromosomes were identified in the turbot at the genome-wide level. Analyses of gene structure, motif composition, and phylogenetic relationships provided valuable data on the HSP70s regarding their evolution, classification, and functional diversity. Expression profiles of the HSP70 genes under five different stresses were investigated by examining multiple RNA-seq datasets. Results showed that 10, 6, 8, 10, and 9 HSP70 genes showed significantly up- or downregulated expression after heat-induced, salinity-induced, and Enteromyxum scophthalmi, Vibrio anguillarum, and Megalocytivirus infection-induced stress, respectively. Among them, hsp70 (hspa1a), hspa1b, and hspa5 showed significant responses to each kind of induced stress, and qPCR analyses further validated their involvement in comprehensive anti-stress, indicating their involvement in organisms’ anti-stress mechanisms. These findings not only provide new insights into the biological function of HSP70s in turbot adapting to various environmental stresses, but also contribute to the development of molecular-based selective breeding programs for the production of stress-resistant turbot strains in the aquaculture industry. Full article

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. Full article

The analytical and diagnostic performances of methods for detecting red sea bream iridovirus (RSIV), which infects marine fish, have not been evaluated. As disease management and transmission control depend on early and reliable pathogen detection, rapid virus detection techniques are crucial. Herein, we evaluated the diagnostic performance of a TaqMan-based real-time polymerase chain reaction (PCR) assay that detects RSIV rapidly and accurately. The assay amplified the RSIV, infectious spleen and kidney necrosis virus, and turbot reddish body iridovirus genotypes of Megalocytivirus and the detection limit was 10.96 copies/reaction. The assay’s performance remained uncompromised even in the presence of nine potential PCR inhibitors, including compounds commonly used in aquaculture. The variation of the cycle threshold values between assays performed by three technicians was evaluated using a plasmid DNA containing the major capsid protein gene sequence. The variation between replicates was low. The diagnostic sensitivity and specificity of the developed assay were evaluated using fish samples (n = 510) and were found to be 100% and 99.60%, respectively. Two technicians evaluated the reproducibility of the assay using fish samples (n = 90), finding a high correlation of 0.998 (p < 0.0001). Therefore, the newly developed real-time PCR assay detects RSIV both accurately and rapidly. Full article

Iridoviruses are an important pathogen of ectothermic vertebrates and are considered a significant threat to aquacultural fish production. Recently, one of the most economically important marine species in China, the large yellow croaker (Larimichthys crocea), has been increasingly reported to be the victim of iridovirus disease. In this study, we isolated and identified a novel iridovirus, LYCIV-ZS-2020, from cage-cultured large yellow croaker farms in Zhoushan island, China. Genome sequencing and subsequent phylogenetic analyses showed that LYCIV-ZS-2020 belongs to the genus Megalocytivirus and is closely related to the Pompano iridoviruses isolated in the Dominican Republic. LYCIV-ZS-2020 enriched from selected tissues of naturally infected large yellow croaker was used in an artificial infection trial and the results proved its pathogenicity in large yellow croaker. This is the first systematic research on the genetic and pathogenic characterization of iridovirus in large yellow croakers, which expanded our knowledge of the iridovirus. Full article

In spring 2019, diseased four-month-old tilapia (Oreochromis spp.) from an aquaculture farm in Southern California, USA were received for diagnostic evaluation with signs of lethargy, anorexia, abnormal swimming, and low-level mortalities. At necropsy, non-specific external lesions were noted including fin erosion, cutaneous melanosis, gill pallor, and coelomic distension. Internal changes included ascites, hepatomegaly, renomegaly, splenomegaly, and multifocal yellow-white nodules in the spleen and kidney. Cultures of spleen and kidney produced bacterial colonies identified as Francisella orientalis. Homogenized samples of gill, brain, liver, spleen, and kidney inoculated onto Mozambique tilapia brain cells (OmB) developed cytopathic effects, characterized by rounding of cells and detaching from the monolayer 6–10 days post-inoculation at 25 °C. Transmission electron microscopy revealed 115.4 ± 5.8 nm icosahedral virions with dense central cores in the cytoplasm of OmB cells. A consensus PCR, targeting the DNA polymerase gene of large double-stranded DNA viruses, performed on cell culture supernatant yielded a sequence consistent with an iridovirus. Phylogenetic analyses based on the concatenated full length major capsid protein and DNA polymerase gene sequences supported the tilapia virus as a novel species within the genus Megalocytivirus, most closely related to scale drop disease virus and European chub iridovirus. An intracoelomic injection challenge in Nile tilapia (O. niloticus) fingerlings resulted in 39% mortality after 16 days. Histopathology revealed necrosis of head kidney and splenic hematopoietic tissues. Full article

Scale drop disease virus (SDDV), an emerging piscine iridovirus prevalent in farmed Asian seabass Lates calcarifer in Southeast Asia, was firstly scientifically descripted in Singapore in 2015. Here, an SDDV isolate ZH-06/20 was isolated by inoculating filtered ascites from diseased juvenile yellowfin seabream into MFF-1 cell. Advanced cytopathic effects were observed 6 days post-inoculation. A transmission electron microscopy examination confirmed that numerous virion particles, about 140 nm in diameter, were observed in infected MFF-1 cell. ZH-06/20 was further purified and both whole genome and virion proteome were determined. The results showed that ZH-06/20 was composed of 131,122 bp with 135 putative viral proteins and 113 of them were further detected by virion proteome. Western blot analysis showed that no (or weak) cross-reaction was observed among several major viral proteins between ZH-06/20 and ISKNV-like megalocytivirus. An artificial challenge showed that ZH-06/20 could cause 100% death to juvenile yellowfin seabream. A typical sign was characterized by severe ascites, but not scale drop, which was considerably different from SDD syndrome in Asian seabass. Collectively, SDDV was confirmed, for the first time, as the causative agent of ascites diseases in farmed yellowfin seabream. Our study offers useful information to better understanding SDDV-associated diseases in farmed fish. Full article

Megalocytivirus is an important viral pathogen to many farmed fishes, including Japanese flounder (Paralichthys olivaceus). In this study, we examined megalocytivirus-induced RNA responses in the spleen of flounder by high-throughput sequencing and integrative analysis of various RNA-seq data. A total of 1327 microRNAs (miRNAs), including 368 novel miRNAs, were identified, among which, 171 (named DEmiRs) exhibited significantly differential expressions during viral infection in a time-dependent manner. For these DEmiRs, 805 differentially expressed target mRNAs (DETmRs) were predicted, whose expressions not only significantly changed after megalocytivirus infection but were also negatively correlated with their paired DEmiRs. Integrative analysis of immune-related DETmRs and their target DEmiRs identified 12 hub DEmiRs, which, together with their corresponding DETmRs, formed an interaction network containing 84 pairs of DEmiR and DETmR. In addition to DETmRs, 19 DEmiRs were also found to regulate six key immune genes (mRNAs) differentially expressed during megalocytivirus infection, and together they formed a network consisting of 21 interactive miRNA-messenger RNA (mRNA) pairs. Further analysis identified 9434 circular RNAs (circRNAs), 169 of which (named DEcircRs) showed time-specific and significantly altered expressions during megalocytivirus infection. Integrated analysis of the DETmR-DEmiR and DEcircR-DEmiR interactions led to the identification of a group of competing endogenous RNAs (ceRNAs) constituted by interacting triplets of circRNA, miRNA, and mRNA involved in antiviral immunity. Together these results indicate that complicated regulatory networks of different types of non-coding RNAs and coding RNAs are involved in megalocytivirus infection. Full article

MicroRNAs (miRNAs) are small non-coding RNAs with important roles in diverse biological processes including immunity. Japanese flounder (Paralichthys olivaceus) is an aquaculture fish species susceptible to the infection of bacterial and viral pathogens including Edwardsiella tarda. In a previous study, pol-miR-novel_547, a novel miRNA of flounder with unknown function, was found to be induced by E. tarda. In the present study, we investigated the regulation and function of pol-miR-novel_547 and its target gene. We found that pol-miR-novel_547 was regulated differently by E. tarda and the viral pathogen megalocytivirus, and pol-miR-novel_547 repressed the expression of PTEN (phosphatase and tensin homolog) of flounder (PoPTEN). PoPTEN is ubiquitously expressed in multiple tissues of flounder and responded to bacterial and viral infections. Interference with PoPTEN expression in flounder cells directly or via pol-miR-novel_547 promoted E. tarda invasion. Consistently, in vivo knockdown of PoPTEN enhanced E. tarda dissemination in flounder tissues, whereas in vivo overexpression of PoPTEN attenuated E. tarda dissemination but facilitated megalocytivirus replication. Further in vitro and in vivo studies showed that PoPTEN affected autophagy activation via the AKT/mTOR pathway and also modulated the process of apoptosis. Together these results reveal for the first time a critical role of fish PTEN and its regulatory miRNA in pathogen infection, autophagy, and apoptosis. Full article

A cross-sectional survey was used to estimate the prevalence of infections with the Infectious spleen and kidney necrosis virus (ISKNV, Megalocytivirus), nervous necrosis virus (NNV, Betanodavirus), and infestations with ectoparasites during the rainy season in juvenile grouper (Epinephelus spp.) farmed in Aceh, Indonesia. The survey was intended to detect aquatic pathogens present at 10% prevalence with 95% confidence, assuming 100% sensitivity and specificity using a sample size of 30 for each diagnostic test. Eight populations of grouper from seven farms were sampled. Additional targeted sampling was conducted for populations experiencing high mortality. Infection with NNV was detected at all farms with seven of the eight populations being positive. The apparent prevalence for NNV ranged from 0% (95% CI: 0–12) to 73% (95% CI: 54–88). All of the fish tested from the targeted samples (Populations 9 and 10) were positive for NNV and all had vacuolation of the brain and retina consistent with viral nervous necrosis (VNN). Coinfections with ISKNV were detected in five populations, with the highest apparent prevalence being 13% (95% CI: 4–31%). Trichodina sp., Cryptocaryon irritans and Gyrodactylus sp. were detected at three farms, with 66% to 100% of fish being infested. Hybrid grouper sourced from a hatchery were 5.4 and 24.9 times more likely to have a NNV infection and a higher parasite load compared to orange-spotted grouper collected from the wild (p < 0.001). This study found that VNN remains a high-impact disease in grouper nurseries in Aceh, Indonesia. Full article