Susceptibility of a number of Australian freshwater fishes to dwarf gourami iridovirus (Infectious spleen and kidney necrosis virus).

Infectious diseases seriously threaten sustainable aquaculture development, resulting in more than $10 billion in economic losses annually. Immersion vaccines are emerging as the key technology for aquatic disease prevention and control. Here, a safe and efficacious candidate immersion vaccine strain (Δorf103r/tk) of infectious spleen and kidney necrosis virus (ISKNV), in which the orf103r and tk genes were knocked out by homologous recombination, is described. Δorf103r/tk was severely attenuated in mandarin fish (Siniperca chuatsi), inducing mild histological lesions, a mortality rate of only 3%, and eliminated within 21 days. A single Δorf103r/tk immersion-administered dose provided long-lasting protection rates over 95% against lethal ISKNV challenge. Δorf103r/tk also robustly stimulated the innate and adaptive immune responses. For example, interferon expression was significantly upregulated, and the production of specific neutralizing antibodies against ISKNV was markedly induced postimmunization. This work provides proof-of-principle evidence for orf103r- and tk-deficient ISKNV for immersion vaccine development to prevent ISKNV disease in aquaculture production. IMPORTANCE Global aquaculture production reached a record of 122.6 million tons in 2020, with a total value of 281.5 billion U.S. dollars (USD). However, approximately 10% of farmed aquatic animal production is lost due to various infectious diseases, resulting in more than 10 billion USD of economic waste every year. Therefore, the development of vaccines to prevent and control aquatic infectious diseases is of great significance. Infectious spleen and kidney necrosis virus (ISKNV) infection occurs in more than 50 species of freshwater and marine fish and has caused great economic losses to the mandarin fish farming industry in China during the past few decades. Thus, it is listed as a certifiable disease by the World Organization for Animal Health (OIE). Herein, a safe and efficient double-gene-deleted live attenuated immersion vaccine against ISKNV was developed, providing an example for the development of aquatic gene-deleted live attenuated immersion vaccine.

Development of a mandarin fish Siniperca chuatsi fry cell line suitable for the study of infectious spleen and kidney necrosis virus (ISKNV)

Bacterial sepsis caused by Aeromonas hydrophila (A. hydrophila) and infectious spleen and kidney necrosis virus disease (ISKNVD) caused by infectious spleen and kidney necrosis virus (ISKNV) frequently result in significant mortality among Chinese perch (Siniperca chuatsi). Co-infection of mandarin fish with A. hydrophila and ISKNV occurs from time to time. In this study, a visual detection method for ISKNV and A. hydrophila was developed, using loop-mediated isothermal amplification (LAMP) and pre-addition of hydroxynaphthol blue. Primers for amplifying LAMP in the same system were designed based on the conserved regions of the MCP gene of infectious spleen and kidney necrosis virus, as well as the hlyA gene of A. hydrophila. The results showed that this method amplified bright trapezoidal bands in the presence of only A. hydrophila or ISKNV and both, with sky blue for positive amplification and violet for negative amplification. There was no cross-reactivity with other pathogens, and fragments of 182 bp, 171 bp and 163 bp appeared after digestion of the A. hydrophila LAMP product and 136 bp, 117 bp and 96 bp appeared after digestion of the ISKNV LAMP product. This holds true even when both positive products are present simultaneously. The minimum detection limit of this method was 100 fg for A. hydrophila and 100 fg for ISKNV, and the minimum detection limit for the mixed template was 1 pg. Overall, this method has high sensitivity and specificity to rapidly detect and distinguish between the two pathogens.

ABSTRACTp53, as an important tumor suppressor protein, has recently been implicated in host antiviral defense. The present study found that the expression of mandarin fish (Siniperca chuatsi) p53 (Sc-p53) was negatively associated with infectious spleen and kidney necrosis virus (ISKNV) and Siniperca chuatsi rhabdovirus (SCRV) proliferation as well as the expression of glutaminase 1 (GLS1) and glutaminolysis pathway-related enzymes glutamate dehydrogenase (GDH) and isocitrate dehydrogenase 2 (IDH2). This indicated that Sc-p53 inhibited the replication and proliferation of ISKNV and SCRV by negatively regulating the glutaminolysis pathway. Moreover, it was confirmed that miR145-5p could inhibit c-Myc expression by targeting the 3′ untranslated region (UTR). Sc-p53 could bind to the miR145-5p promoter region to promote its expression and to further inhibit the expression of c-Myc. The expression of c-Myc was proved to be positively correlated with the expression of GLS1 as well. All these suggested a negative relationship between the Sc-p53/miR145-5p/c-Myc pathway and GLS1 expression and glutaminolysis. However, it was found that after ISKNV and SCRV infection, the expressions of Sc-p53, miR145-5p, c-Myc, and GLS1 were all significantly upregulated, which did not match the pattern in normal cells. Based on the results, it was suggested that ISKNV and SCRV infection altered the Sc-p53/miR145-5p/c-Myc pathway. All of above results will provide potential targets for the development of new therapeutic strategies against ISKNV and SCRV.IMPORTANCE Infectious spleen and kidney necrosis virus (ISKNV) and Siniperca chuatsi rhabdovirus (SCRV) as major causative agents have caused a serious threat to the mandarin fish farming industry (J.-J. Tao, J.-F. Gui, and Q.-Y. Zhang, Aquaculture 262:1–9, 2007, https://doi.org/10.1016/j.aquaculture.2006.09.030). Viruses have evolved the strategy to shape host-cell metabolism for their replication (S. K. Thaker, J. Ch’ng, and H. R. Christofk, BMC Biol 17:59, 2019, https://doi.org/10.1186/s12915-019-0678-9). Our previous studies showed that ISKNV replication induced glutamine metabolism reprogramming and that glutaminolysis was required for efficient replication of ISKNV and SCRV. In the present study, the mechanistic link between the p53/miR145-5p/c-Myc pathway and glutaminolysis in the Chinese perch brain (CPB) cells was provided, which will provide novel insights into ISKNV and SCRV pathogenesis and antiviral treatment strategies.

Infectious spleen and kidney necrosis virus (ISKNV) is a species within the genus Megalocytivirus (family Iridoviridae), which causes high mortality disease in many freshwater and marine fish species. ISKNV was first reported in Asia and is an emerging threat to aquaculture with increasing global distribution, in part due to its presence in ornamental fish with clinical and subclinical infections. The species ISKNV includes three genotypes: red seabream iridovirus (RSIV), turbot reddish body iridovirus (TRBIV), and ISKNV. There is an increasing overlap in the recognized range of susceptible fish hosts and the geographic distribution of these distinct genotypes. To better understand the disease caused by ISKNV, a nucleic acid hybridization capture enrichment was used prior to sequencing to characterize whole genomes from archived clinical specimens of aquaculture and ornamental fish from Southeast Asia (n = 16). The method was suitable for tissue samples containing 2.50 × 104-4.58 × 109 ISKNV genome copies mg-1. Genome sequences determined using the hybridization capture method were identical to those obtained directly from tissues when there was sufficient viral DNA to sequence without enrichment (n = 2). ISKNV genomes from diverse locations, environments, and hosts had very high similarity and matched established genotype classifications (14 ISKNV genotype Clade 1 genomes with >98.81% nucleotide similarity). Conversely, two different genotypes were obtained at the same time and location (RSIV and ISKNV from grouper, Indonesia with 92.44% nucleotide similarity). Gene-by-gene analysis with representative ISKNV genomes identified 59 core genes within the species (>95% amino acid identity). The 14 Clade 1 ISKNV genomes in this study had 100% aa identity for 92-105 of 122 predicted genes. Despite high overall sequence similarity, phylogenetic analyses using single nucleotide polymorphisms differentiated isolates from different host species, country of origin, and time of collection. Whole genome studies of ISKNV and other megalocytiviruses enable genomic epidemiology and will provide information to enhance disease control in aquaculture.

Efficacy of a formalin-killed cell vaccine against infectious spleen and kidney necrosis virus (ISKNV) and immunoproteomic analysis of its major immunogenic proteins

Development and application of a sensitive droplet digital PCR (ddPCR) for the detection of infectious spleen and kidney necrosis virus

Infectious spleen and kidney necrosis virus (ISKNV) is a fish-pathogenic virus belonging to the genus Megalocytivirus of the family Iridoviridae. In 2018, disease occurrences (40-50% cumulative mortality) associated with ISKNV infection were reported in grown-out Asian sea bass (Lates calcarifer) cultured in an inland freshwater system in Thailand. Clinical samples were collected from seven distinct farms located in the eastern and central regions of Thailand. The moribund fish showed various abnormal signs, including lethargy, pale gills, darkened body, and skin hemorrhage, while hypertrophied basophilic cells were observed microscopically in gill, liver, and kidney tissue. ISKNV infection was confirmed on six out of seven farms using virus-specific semi-nested PCR. The MCP and ATPase genes showed 100% sequence identity among the virus isolates, and the virus was found to belong to the ISKNV genotype I clade. Koch's postulates were later confirmed by challenge assay, and the mortality of the experimentally infected fish at 21 days post-challenge was 50-90%, depending on the challenge dose. The complete genome of two ISKNV isolates, namely KU1 and KU2, was recovered directly from the infected specimens using a shotgun metagenomics approach. The genome length of ISKNV KU1 and KU2 was 111,487 and 111,610 bp, respectively. In comparison to closely related ISKNV strains, KU1 and KU2 contained nine unique genes, including a caspase-recruitment-domain-containing protein that is potentially involved in inhibition of apoptosis. Collectively, this study indicated that inland cultured Asian sea bass are infected by homologous ISKNV strains. This indicates that ISKNV genotype I should be prioritized for future vaccine research.

Infectious spleen and kidney necrosis virus (ISKNV), a member of the genus Megalocytivirus in the family Iridoviridae, can infect over 50 fish species and cause significant economic losses in Asia. Our previous study showed that hypoxia triggers the hypoxia-inducible factor pathway (HIF-pathway), leading to increased replication of ISKNV through promoting the upregulation of viral hypoxic response genes like orf077r. This study delved into the molecular mechanism of how ISKNV manipulates the HIF-pathway to enhance its replication. In vitro and in vivo experiments confirmed that ISKNV infection activated the HIF-pathway, which in turn promoted ISKNV replication. These findings suggest that ISKNV actively manipulates the HIF-pathway. Co-immunoprecipitation experiments revealed that the ISKNV-encoded protein VP077R interacts with the Von Hippel−Lindau (VHL) protein at the HIF-binding region, competitively inhibiting the interaction of HIF-1α with VHL. This prevents HIF degradation and activates the HIF-pathway. Furthermore, VP077R interacts with factor-inhibiting HIF (FIH), recruiting FIH and S-phase kinase-associated protein 1 (Skp1) to form an FIH – VP077R – Skp1 complex. This complex promotes FIH protein degradation via ubiquitination, further activating the HIF-pathway. These findings indicated that ISKNV takes over the HIF-pathway by releasing two “brakes” on this pathway (VHL and FIH) via VP077R, facilitating virus replication. We speculate that hypoxia initiates a positive feedback loop between ISKNV VP077R and the HIF pathway, leading to the outbreak of ISKNV disease. This work offers valuable insights into the complex interactions between the environment, host, and virus.

Antigenic identification of virion structural proteins from infectious spleen and kidney necrosis virus

Multiplex qPCR development for the simultaneous and rapid detection of largemouth bass virus and infectious spleen and kidney necrosis virus in aquaculture

The infectious spleen and kidney necrosis virus (ISKNV) is a highly lethal virus, which has brought significant losses to aquaculture. Therefore, a new vaccine against ISKNV with high efficiency, safety and convenience must be developed. While baculoviruses are more commonly used as protein expression systems for vaccine antigen production, this paper used baculovirus technology to develop a live-vector vaccine, BacMCP, which contains the coding sequence of the major capsid protein (MCP) (GenBank accession no. AF371960) of ISKNV and is driven by a CMV promoter. Real-time PCR and immunofluorescence showed that the MCP gene was successfully delivered to and expressed in fish cells and tissues inoculated with BacMCP. Immune-related gene (IgM, TGF-β, IL-1, IL-8, TNF-α) expression was induced in BacMCP-treated groups of largemouth bass compared with control groups. Specific antibodies could be detected in the serum of BacMCP injection-vaccinated largemouth bass by ELISA. After injection or immersion vaccination with BacMCP for 21 days, largemouth bass were infected with ISKNV. The immune effect of the injected immunization on fish in different sizes was evaluated. The vaccine efficacy of injection-vaccinated bass was 100% in small bass and 85.7% in large bass. The vaccine efficacy of immersion-vaccinated small bass was 77.3%. This study suggested that BacMCP can be used as a vector-based vaccine candidate to prevent the diseases caused by ISKNV infection.

Infectious spleen and kidney necrosis virus (ISKNV) is the causative agent of a fatal disease in many fish species, resulting in mass mortalities and significant economic losses. Since its introduction to Ghana in late 2018 and in the absence of effective vaccines, the crude practice of heat-shock treatment (HST) on deliberately exposed cultured tilapia fingerlings was widely adopted by farmers to control the disease in Ghana with some apparent success. This study investigated the interplay between the expression of heat-shock proteins (HSPs) and viral replication during ISKNV infection. An in vitro experimental challenge study which involved deliberate infection with ISKNV and subsequent exposure of primary Oreochromis niloticus (tilapia) brain cell lines to HST at 48 hours post-infection was carried out. The ISKNV was confirmed by Oxford Nanopore Sequencing of the full major capsid protein (MCP), while the species identity of the cell line was confirmed by Sanger sequencing of the cytochrome C oxidase (COX1) genomic region. The test groups and control groups were screened at various time points for viral proliferation and HSP marker expression using quantitative PCR (qPCR). Exposure to heat shock significantly increased HSP 90 and 47 expressions by fourfold and sixfold, respectively, with a concomitant 10-fold decrease in viral load as compared to the non-heat-shock group. Viral apoptosis gene ORF 005L was significantly downregulated following increase in HSPs expression. This initial finding implies that HST may play an important role in suppressing viral replication through the apoptosis regulatory gene ORF 005L. This information will contribute to the understanding of the beneficial effect of heat-shock therapy used in control of the viral pathogen in aquaculture. Further studies in controlled in vivo experiments will give more clarity to the general effect of this treatment on tilapia growth and ISKNV persistence in infected fish populations.IMPORTANCEInfection of tilapia by infectious spleen and kidney necrosis virus (ISKNV) is trending toward endemicity in Ghanaian lake-based tilapia farms, and it is an important fish pathogen, worldwide. This study provides a potential mechanism to explain the reported role of heat-shock in protecting fish from the negative effects of ISKNV infection. Thus, it offers strong evidence for heat-shock therapy and will lead to better disease management in Ghana and worldwide. Additionally, there are other research avenues that may lead to some therapeutic options down the line.

Viral outbreaks are a constant threat to aquaculture, limiting production for better global food security. A lack of diagnostic testing and monitoring in resource-limited areas hinders the capacity to respond rapidly to disease outbreaks and to prevent viral pathogens becoming endemic in fisheries productive waters. Recent developments in diagnostic testing for emerging viruses, however, offers a solution for rapid in situ monitoring of viral outbreaks. Genomic epidemiology has furthermore proven highly effective in detecting viral mutations involved in pathogenesis and assisting in resolving chains of transmission. Here, we demonstrate the application of an in-field epidemiological tool kit to track viral outbreaks in aquaculture on farms with reduced access to diagnostic labs, and with non-destructive sampling. Inspired by the "lab in a suitcase" approach used for genomic surveillance of human viral pathogens and wastewater monitoring of COVID19, we evaluated the feasibility of real-time genome sequencing surveillance of the fish pathogen, Infectious spleen and kidney necrosis virus (ISKNV) in Lake Volta. Viral fractions from water samples collected from cages holding Nile tilapia (Oreochromis niloticus) with suspected ongoing ISKNV infections were concentrated and used as a template for whole genome sequencing, using a previously developed tiled PCR method for ISKNV. Mutations in ISKNV in samples collected from the water surrounding the cages matched those collected from infected caged fish, illustrating that water samples can be used for detecting predominant ISKNV variants in an ongoing outbreak. This approach allows for the detection of ISKNV and tracking of the dynamics of variant frequencies, and may thus assist in guiding control measures for the rapid isolation and quarantine of infected farms and facilities.

Deletion of the Infectious spleen and kidney necrosis virus ORF069L reduces virulence to mandarin fish Siniperca chuatsi