Tongyan Lu

Infectious hematopoietic necrosis virus (IHNV), a salmonid rhabdovirus, causes severe mortality exceeding 90% in both wild and farmed salmon and trout. Frequent outbreaks of IHNV highlight the urgent need for rapid detection methods to support effective prevention and control. This study developed a double-antibody sandwich ELISA (DAS-ELISA) targeting the nucleocapsid (N) protein of IHNV. Two peptides derived from the N protein—selected for their strong antigenicity, high level of conservation, and surface accessibility—were used as immunogens to generate two specific monoclonal antibodies. Following optimization, the DAS-ELISA was established using monoclonal antibody N-15 as the capture antibody and horseradish peroxidase (HRP)-conjugated antibody N-106 as the detection antibody. The results of this study demonstrated that DAS-ELISA exhibited high specificity for multiple IHNV strains and showed no cross-reactivity with IPNV, SVCV, or VHSV. The detection sensitivity of DAS-ELISA for IHNV was determined to be 10³ TCID₅₀/mL. Parallel analysis of 293 clinical samples using DAS-ELISA and WOAH reference method demonstrated a concordance rate of 92.83% (κ = 0.856). These results confirm that the established DAS-ELISA exhibits high sensitivity, specificity, broad-spectrum applicability, and repeatability. In conclusion, this DAS-ELISA provides a reliable and efficient tool for high-throughput early detection of IHNV infection in clinical settings.

The journal retracts the article “Andrographolide Alleviates Oxidative Damage and Inhibits Apoptosis Induced by IHNV Infection via CTSK/BCL2/Cytc Axis”

The rainbow trout (Oncorhynchus mykiss) is an important commercial fish. Studies have shown that probiotics can promote the healthy growth of fish. In this study, we assessed the impact of an endogenous Bacillus subtilis strain (RT-BS07) on the growth, survival and immunological response of juvenile rainbow trout. Additionally, the morphology of rainbow trout’s intestinal tissue was measured. Control (CT) and experimental groups (B) were fed with conventional and probiotic-supplemented diets (1.0 × 10⁸ CFU per gram) for 42 days, respectively. Following feeding, a challenge experiment was carried out with Aeromonas hydrophila at 1.0 × 10⁷ CFU/mL to determine the experimental fish’s tolerance to the bacteria. The results showed that the weight gain rate (WGR) in group B was 118.2%, which was significantly higher than that in group CT (54.86%) after 42 days. The height of intestinal villi and the number of goblet cells increased in group B, thus increasing the resistance of rainbow trout to pathogenic bacteria. The alkaline phosphatase (AKP) activity (U/mg prot) in the liver, spleen and intestinal tissues of group B was significantly higher than that in the CT group. The expression of interleukin-1β (IL-1β) in the intestinal tissue of group B was 6-fold higher than that in group CT. Furthermore, the expression of growth hormone-2 (GH-2) in the spleen was 3-fold higher than that in group CT. After 12 days of A. hydrophila injection, the survival rate was 40% and 70% in group CT and group B, respectively. Collectively, the present study demonstrated that the addition of endogenous B. subtilis strain (1.0 × 10⁸ CFU per gram) to the feed can effectively promote the growth of rainbow trout and enhance immunity, which is beneficial to rainbow trout culture.

Infectious hematopoietic necrosis virus (IHNV) causes infectious hematopoietic necrosis and severe economic losses to salmon and trout aquaculture worldwide. Currently, the only commercial vaccine against IHNV is a DNA vaccine with some biosafety concerns. Hence, more effective vaccines and antiviral drugs are needed to prevent IHNV infection. In this study, 1,483 compounds were screened from a traditional Chinese medicine monomer library, and bufalin showed potential antiviral activity against IHNV. The 50% cytotoxic concentration of bufalin was >20 µM, and the 50% inhibitory concentration was 0.1223 µΜ against IHNV. Bufalin showed the inhibition of diverse IHNV strains in vitro , which confirmed that it had an inhibitory effect against all IHNV strains, rather than random activity against a single strain. The bufalin-mediated block of IHNV infection occurred at the viral attachment and RNA replication stages, but not internalization. Bufalin also inhibited IHNV infection in vivo and significantly increased the survival of rainbow trout compared with the mock drug-treated group, and this was confirmed by in vivo viral load monitoring. Our data showed that the anti-IHNV activity of bufalin was proportional to extracellular Na ⁺ concentration and inversely proportional to extracellular K ⁺ concentration, and bufalin may inhibit IHNV infection by targeting Na ⁺ /K ⁺ -ATPase. The in vitro and in vivo studies showed that bufalin significantly inhibited IHNV infection and may be a promising candidate drug against the disease in rainbow trout. IMPORTANCE: Infectious hematopoietic necrosis virus (IHNV) is the pathogen of infectious hematopoietic necrosis (IHN) which outbreak often causes huge economic losses and hampers the healthy development of salmon and trout farming. Currently, there is only one approved DNA vaccine for IHN worldwide, but it faces some biosafety problems. Hence, more effective vaccines and antiviral drugs are needed to prevent IHNV infection. In this study, we report that bufalin, a traditional Chinese medicine, shows potential antiviral activity against IHNV both in vitro and in vivo . The bufalin-mediated block of IHNV infection occurred at the viral attachment and RNA replication stages, but not internalization, and bufalin inhibited IHNV infection by targeting Na ⁺ /K ⁺ -ATPase. The in vitro and in vivo studies showed that bufalin significantly inhibited IHNV infection and may be a promising candidate drug against the disease in rainbow trout.

Infectious hematopoietic necrosis virus (IHNV) is an important pathogen that causes significant economic losses to salmon trout farming. Although vaccines have been invented for the treatment of IHNV, findings from our previous survey show that breeding enterprises and farmers require effective oral drugs or immune enhancers. However, studies on the development of oral drugs are limited. In the present study, we used bioinformatics methods to predict the protein targets of andrographolide (Andro) in IHNV. Cells were infected with IHNV, and the effect of andrographolide was explored by evaluating the expression levels of genes implicated in oxidative stress, activities of antioxidant enzymes, and the expression of genes implicated in apoptosis and necrosis. In the present study, cells were divided into NC, IHNV, IHNV+10 μM andrographolide, and IHNV+20 μM andrographolide groups. qRT-PCR was performed to determine the expression level of genes, and an antioxidant enzyme detection kit was used to evaluate the activities of antioxidant enzymes. Fluorescent staining was performed using a reactive oxygen species detection kit (ROS) and Hoechst 33342/PI double staining kit, and the mechanism of alleviation of apoptosis and oxidative stress andrographolide after IHNV infection was determined. The results indicated that andrographolide inhibits viral growth by binding to the NV protein of IHNV and increasing the antioxidant capacity of the body through the CTSK/BCL2/Cytc axis, thereby inhibiting the occurrence of IHNV-induced apoptosis. This is the first study to explore the antagonistic mechanism of action of andrographolide in alleviating IHNV infection. The results provide valuable information on alternative strategies for the treatment of IHNV infection during salmon family and provide a reference for the use of andrographolide as an antioxidant agent in agricultural settings.