Low-temperature immunization attenuates the residual virulence of orf074r gene-deleted infectious spleen and kidney necrosis virus: a candidate immersion vaccine.

IMPORTANCE: Global aquaculture production yielded a record of 122.9 million tons in 2022. However, ~10% of farmed aquatic animal production is lost each year due to various infectious diseases, resulting in substantial economic waste. Therefore, the development of vaccines is important for the prevention and control of aquatic infectious diseases. Gene-deletion live attenuated vaccines are efficacious because they mimic natural pathogen infection and generate a strong antibody response, thus showing good potential for administration via immersion. However, most gene-deletion viruses still have residual virulence, and thus, gene-deletion immersion vaccines for aquatic viruses are rarely developed. In this study, an orf074r deletion strain (Δorf074r) of ISKNV with residual virulence was constructed, and an immunization process was developed to reduce its residual virulence at 22°C, thereby making it a potential immersion vaccine against ISKNV. Our work will aid in the development of an aquatic gene-deletion live-attenuated immersion vaccine.

A Safe and Efficient Double-Gene-Deleted Live Attenuated Immersion Vaccine to Prevent the Disease Caused by the 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.

Interaction of Teleost Fish TRPV4 with DEAD Box RNA Helicase 1 Regulates Iridovirus Replication.

Viral diseases are a significant risk to the aquaculture industry. Transient receptor potential vanilloid 4 (TRPV4) has been reported to be involved in regulating viral activity in mammals, but its regulatory effect on viruses in teleost fish remains unknown. Here, the role of the TRPV4-DEAD box RNA helicase 1 (DDX1) axis in viral infection was investigated in mandarin fish (Siniperca chuatsi). Our results showed that TRPV4 activation mediates Ca2+ influx and facilitates infectious spleen and kidney necrosis virus (ISKNV) replication, whereas this promotion was nearly eliminated by an M709D mutation in TRPV4, a channel Ca2+ permeability mutant. The concentration of cellular Ca2+ increased during ISKNV infection, and Ca2+ was critical for viral replication. TRPV4 interacted with DDX1, and the interaction was mediated primarily by the N-terminal domain (NTD) of TRPV4 and the C-terminal domain (CTD) of DDX1. This interaction was attenuated by TRPV4 activation, thereby enhancing ISKNV replication. DDX1 could bind to viral mRNAs and facilitate ISKNV replication, which required the ATPase/helicase activity of DDX1. Furthermore, the TRPV4-DDX1 axis was verified to regulate herpes simplex virus 1 replication in mammalian cells. These results suggested that the TRPV4-DDX1 axis plays an important role in viral replication. Our work provides a novel molecular mechanism for host involvement in viral regulation, which would be of benefit for new insights into the prevention and control of aquaculture diseases. IMPORTANCE In 2020, global aquaculture production reached a record of 122.6 million tons, with a total value of $281.5 billion. Meanwhile, frequent outbreaks of viral diseases have occurred in aquaculture, and about 10% of farmed aquatic animal production has been lost to infectious diseases, resulting in more than $10 billion in economic losses every year. Therefore, an understanding of the potential molecular mechanism of how aquatic organisms respond to and regulate viral replication is of great significance. Our study suggested that TRPV4 enables Ca2+ influx and interactions with DDX1 to collectively promote ISKNV replication, providing novel insights into the roles of the TRPV4-DDX1 axis in regulating the proviral effect of DDX1. This advances our understanding of viral disease outbreaks and would be of benefit for studies on preventing aquatic viral diseases.

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

Mandarin fish (Siniperca chuatsi) is a significant cultured species with high added value in China. With the expansion of farming, diseases of mandarin fish such as Infectious spleen and kidney necrosis virus (ISKNV) diseases are becoming more and more serious. Human endogenous retrovirus subfamily H long terminal repeat associating protein 2 (HHLA2) is a type 1 transmembrane molecule with three extracellular Ig domains (IgV-IgC-IgV) and plays important roles in the T cell proliferation and tumorigenesis. The HHLA2-homologues have not been found in virus. In this study, a viral HHLA2 protein encoded by ISKNV ORF069L was identified and the virulence of the deleted ORF069L reconstruction ISKNV strain (ΔORF069L) was investigated. ISKNV ORF069L gene was predicted to encode a 222-amino acids peptide. The bioinformation analysis revealed that ISKNV ORF069L contained an Ig HHLA2 domain and was homologous to vertebrate B7-CD28 family proteins. The recombinant virus strain of ΔORF069L was constructed by homologous recombination technology. The virus titer and growth curves between ISKNV wild type (WT) and ΔORF069L on cellular level showed no significant differences indicating that the ORF069L did not influence the ISKNV replication. The expression levels of immune-related genes (Mx1, IL-1ß, IL-8, TNF-a and IgM) were increased in fish infected with ΔORF069L, compared to those in fish infected with ISKNV WT. Furthermore, the lethality caused by ΔORF069L declined by 40% compared with ISKNV WT, indicating that ORF069L was a virulence gene of ISKNV. Most importantly, the protection rate was nearly 100% for fish immunized with ΔORF069L strain. Those results suggested that ΔORF069L could be developed as a potential attenuated vaccine against ISKNV. Our work will be beneficial to promote the development of gene deletion attenuated vaccines for ISKNV disease.

Evaluation of a Low-Temperature Immersion Immunization Strategy for the Infectious Spleen and Kidney Necrosis Virus orf037l Gene-Deleted Attenuated Vaccine.

BACKGROUND: Infectious spleen and kidney necrosis virus (ISKNV) poses a significant threat to aquaculture sustainability, particularly affecting mandarin fish (Siniperca chuatsi) and causing significant economic losses. METHODS: To address this challenge, this study developed an ISKNV Δorf037l vaccine strain, where the orf037l gene was knocked out. Infection assays conducted at 28 °C showed that the knocking out the orf037l gene decreased the virulence of ISKNV and reduced lethality against mandarin fish by 26.7% compared to wild-type ISKNV. To further diminish residual virulence, the effect of low-temperature (22 °C) immersion immunization was evaluated. RESULTS: The results indicate that low temperature significantly diminished the virulence of the Δorf037l vaccine strain, elevating the survival rate of mandarin fish to 90%. Furthermore, the vaccine strain effectively triggered the expression of crucial immune-related genes, such as IFN-h, IL-1, IκB, Mx, TNF-α, and Viperin, while inducing the production of specific neutralizing antibodies. Low-temperature immersion with Δorf037l achieved a high relative percentage of survival of 92.6% (n = 30) in mandarin fish, suggesting the potential of Δorf037l as a promising immersion vaccine candidate. CONCLUSIONS: These findings contribute to advancing fish immersion vaccine development and demonstrate the importance and broad applicability of temperature optimization strategies in vaccine development. Our work carries profound implications for both the theoretical understanding and practical application in aquaculture disease control.

Hypermethylated genome of a fish vertebrate iridovirus ISKNV plays important roles in viral infection.

Iridoviruses are nucleocytoplasmic large dsDNA viruses that infect invertebrates and ectothermic vertebrates. The hypermethylated genome of vertebrate iridoviruses is unique among animal viruses. However, the map and function of iridovirus genomic methylation remain unknown. Herein, the methylated genome of Infectious spleen and kidney necrosis virus (ISKNV, a fish iridovirus), and its role in viral infection, are investigated. The methylation level of ISKNV is 23.44%. The hypermethylated genome is essential for ISKNV amplification, but there is no correlation between hypermethylation and viral gene expression. The hypomethylated ISKNV (obtained via 5-Azacytidine) activates a strong immunoreaction in vitro and reduces its pathogenicity in vivo. The unmethylated viral DNA can induce a stronger immunoreaction in vitro, whereas inactivated hypomethylated ISKNV can induce a stronger immunoreaction in vivo, suggesting ISKNV may evade from immune system by increasing its genome methylation level. Our work provides new insights into the role of genome methylation in viral infection.

Conceptual Model, Experiment and Numerical Simulation of Diaphragm Wall Leakage Detection Using Distributed Optical Fiber.

Leakage in the diaphragm wall is difficult to detect in deep foundation pits. In this study, the conceptual model of active and passive thermal leak detection methods was proposed according to the occurrence of temperature field anomalies caused by seepage. Experiments were performed using a heating system and an optical fiber temperature measurement system to verify the thermal leakage detection systems. Numerical simulations were performed to understand the mechanism of the detecting method. Results indicated that the optical cable could detect the low-temperature anomaly in the active temperature field leak detection. The arrangement method of the leakage detection system was also presented in actual engineering.

Mechanism of Detecting the Construction Quality of a Diaphragm Wall by an Infrared Thermal Field and Engineering Application.

During underground space exploitation in the urbanization process, numerous foundation pits were constructed where a diaphragm wall was often used as a retaining structure and waterproof curtain. Due to complicated engineering geological conditions or improper construction, diaphragm walls and wall joints often exhibit quality defects. Groundwater leaked from these quality defects to foundation pits during excavation, endangering the safety of the pit and surrounding facilities. The current leakage identification of the underground retaining structure was performed by artificial visual detection, which cannot satisfy the engineering requirement. The temperature field in the leakage area of the diaphragm wall was different from other areas. The leakage wall imaging system using a thermal imager was efficient in visualizing leaking, which was not visible to the naked eye. In this study, infrared thermal imaging technology was introduced in potential leakage detection for the diaphragm wall of a foundation pit. The infrared radiation characteristics of the diaphragm wall leakage and the potential leakage parts were studied through laboratory simulation tests and on-site detection methods. The maximum temperature appeared at the water outlet and the surface of the defect with hidden defect, and the temperature field was symmetrically distributed along the cross-section direction. In the potential leakage area, the temperature difference at the penetration point was 23.4 °C when the initial water pressure was 10 kPa. The temperature difference at the penetration point was 21.8 °C when the initial water pressure was 30 kPa. In the field test, the maximum temperature difference between the leakage area and the surrounding wall was 4.5 °C. The study can provide a reference for similar engineering.

Numerical simulation of foundation pit dewatering using horizontal seepage reducing body.

Groundwater level has to be lowered during deep excavation. A vertical curtain is usually adopted to control the drawdown inside and outside a foundation pit in a built-up area. However, the cost and working difficulty increases substantially with the rise in depth of vertical curtains. In the manuscript, a man-made horizontal seepage reducing body (HSRB) was introduced to shorten the vertical curtain depth and control drawdown. With the No. 4 shaft foundation pit of Guangyuan Project, Shanghai as background, HSRB was proposed in foundation pit dewatering. Microbially induced carbonate precipitation grouting technology was recommended to form an environment-friendly HSRB. Numerical method was used to simulate and understand the influence of position, thickness, and hydraulic conductivity of HSRB on groundwater level. The non-separated HSRB was better than the separate HSRB. Decreasing HSRB hydraulic conductivity was better than increasing HSRB depth. Four seepage modes were summarized considering vertical curtain penetration conditions into multi-aquifer, and the fifth seepage mode was formed for vertical curtain using man-made HSRB, which can be referred by similar engineering applications.

The Interaction of Mandarin Fish DDX41 with STING Evokes type I Interferon Responses Inhibiting Ranavirus Replication.

DDX41 is an intracellular DNA sensor that evokes type I interferon (IFN-I) production via the adaptor stimulator of interferon gene (STING), triggering innate immune responses against viral infection. However, the regulatory mechanism of the DDX41-STING pathway in teleost fish remains unclear. The mandarin fish (Siniperca chuatsi) is a cultured freshwater fish species that is popular in China because of its high market value. With the development of a high-density cultural mode in mandarin fish, viral diseases have increased and seriously restricted the development of aquaculture, such as ranavirus and rhabdovirus. Herein, the role of mandarin fish DDX41 (scDDX41) and its DEAD and HELIC domains in the antiviral innate immune response were investigated. The level of scDDX41 expression was up-regulated following treatment with poly(dA:dT) or Mandarin fish ranavirus (MRV), suggesting that scDDX41 might be involved in fish innate immunity. The overexpression of scDDX41 significantly increased the expression levels of IFN-I, ISGs, and pro-inflammatory cytokine genes. Co-immunoprecipitation and pull-down assays showed that the DEAD domain of scDDX41 recognized the IFN stimulatory DNA and interacted with STING to activate IFN-I signaling pathway. Interestingly, the HELIC domain of scDDX41 could directly interact with the N-terminal of STING to induce the expression levels of IFN-I and ISGs genes. Furthermore, the scDDX41 could enhance the scSTING-induced IFN-I immune response and significantly inhibit MRV replication. Our work would be beneficial to understand the roles of teleost fish DDX41 in the antiviral innate immune response.

Predictions and mitigation strategies of PM2.5 concentration in the Yangtze River Delta of China based on a novel nonlinear seasonal grey model.

High delicate particulate matter (PM2.5) concentration can seriously reduce air quality, destroy the environment, and even jeopardize human health. Accordingly, accurate prediction for PM2.5 plays a vital role in taking precautions against upcoming air ambient pollution incidents. However, due to the disturbance of seasonal and nonlinear characteristics in the raw series, pronounced forecasts are confronted with tremendous handicaps, even though for seasonal grey prediction models in the preceding researches. A novel seasonal nonlinear grey model is initially designed to address such issues by integrating the seasonal adjustment factor, the conventional Weibull Bernoulli grey model, and the cultural algorithm, simultaneously depicting the seasonality and nonlinearity of the original data. Experimental results from PM2.5 forecasting of four major cities (Shanghai, Nanjing, Hangzhou, and Hefei) in the YRD validate that the proposed model can obtain more accurate predictive results and stronger robustness, in comparison with grey prediction models (SNGBM(1,1) and SGM(1,1)), conventional econometric technology (SARIMA), and machine learning methods (LSSVM and BPNN) by employing accuracy levels. Finally, the future PM2.5 concentration is forecasted from 2020 to 2022 using the proposed model, which provides early warning information for policy-makers to develop PM2.5 alleviation strategies.

Development of a gene-deleted live attenuated candidate vaccine against fish virus (ISKNV) with low pathogenicity and high protection.

Aquaculture provides important food, nutrition, and income sources for humans. However, aquaculture industry is seriously threatened by viral diseases. Infectious spleen and kidney necrosis virus (ISKNV) disease causes high mortality and economic losses to the fish culture industry in Asia and has been listed as a certifiable disease by the International Epizootic Office. Vaccine development is urgent to control this disease. Here, a gene-deleted live attenuated candidate vaccine (ΔORF022L) against ISKNV with low pathogenicity and high protection was developed. ΔORF022L replicated well in mandarin fish fry-1 cells and showed similar structure with wild-type ISKNV. However, the pathogenicity was significantly lower as 98% of the mandarin fish infected with ΔORF022L survived, whereas all those infected with wild-type ISKNV died. Of importance, 100% of the ΔORF022L-infected fish survived the ISKNV challenge. ΔORF022L induced anti-ISKNV specific antibody response and upregulation of immune-related genes. This work could be beneficial to the control of fish diseases.