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This study presents the development of a quantitative evaluation method utilizing machine vision technology to characterize the extent of body surface damage in largemouth bass (Micropterus salmoides) infected with largemouth bass ranavirus (LMBV). High-resolution, multi-angle images (6000 × 4000 pixels) of the body surface from 239 infected specimens were acquired at a fixed distance of 40 cm using a SONY ILCE-7RM3 digital camera within a GODOX-LST60 softbox. Key parameters, including the number of segmented injury areas, the count of body surface lesions, and the total lesion area, were analyzed. These parameters were integrated through principal component analysis (PCA) to construct a comprehensive damage scoring model. The severity of viral-induced body surface damage was categorized into four grades: uninjured (0), minor injury (1), moderate injury (2), and severe injury (3). Histopathological examination revealed that early-stage infection (grade 1) predominantly exhibited localized hemorrhagic spots in the muscular region of the body side (B/E region) with limited lesion area. In contrast, moderate to severe infections (grades 2–3) were characterized by extensive ulceration, muscle necrosis, and visceral lesions, including hepatic fibrosis and splenic granulomatous formations. Quantitative real-time PCR (qRT-PCR) analysis demonstrated a progressive upregulation of pro-inflammatory cytokines (IL-6, IL-8, TNF-α, CXCL2) in immune organs, concomitant with increased expression of apoptosis-related genes (CASP8, CYC). This study successfully established a rapid and objective quantitative grading system for ranavirus infection, offering a novel technical approach for early diagnosis and precise prevention and control strategies against largemouth bass ranavirus.

Largemouth bass virus (LMBV), belonging to the genus Ranavirus, causes high mortality and heavy economic losses in largemouth bass aquaculture. In the present study, a novel cell line, designated as MsF, was established from the fin of largemouth bass (Micropterus salmoides), and applied to investigate the characteristics of cell death induced by LMBV. MsF cells showed susceptibility to LMBV, evidenced by the occurrence of a cytopathic effect (CPE), increased viral gene transcription, protein synthesis, and viral titers. In LMBV-infected MsF cells, two or more virus assembly sites were observed around the nucleus. Notably, no apoptotic bodies occurred in LMBV-infected MsF cells after nucleus staining, suggesting that cell death induced by LMBV in host cells was distinct from apoptosis. Consistently, DNA fragmentation was not detected in LMBV-infected MsF cells. Furthermore, only caspase-8 and caspase-3 were significantly activated in LMBV-infected MsF cells, suggesting that caspases were involved in non-apoptotic cell death induced by LMBV in host cells. In addition, the disruption of the mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) generation were detected in both LMBV-infected MsF cells and fathead minnow (FHM) cells. Combined with our previous study, we propose that cell death induced by LMBV infection was cell type dependent. Although LMBV-infected MsF cells showed the characteristics of non-apoptotic cell death, the signal pathways might crosstalk and interconnect between apoptosis and other PCD during LMBV infection. Together, our results not only established the in vitro LMBV infection model for the study of the interaction between LMBV and host cells but also shed new insights into the mechanisms of ranavirus pathogenesis.

The disease caused by Largemouth bass ranavirus (LMBV) is one of the most severe viral diseases in largemouth bass (Micropterus salmoides). It is crucial to evaluate the genetic resistance of largemouth bass to LMBV and develop markers for disease-resistance breeding. In this study, 100 individuals (45 resistant and 55 susceptible) were sequenced and evaluated for resistance to LMBV and a total of 2,579,770 variant sites (SNPs-single-nucleotide polymorphisms (SNPs) and insertions–deletions (InDels)) were identified. A total of 2348 SNPs-InDels and 1018 putative candidate genes associated with LMBV resistance were identified by genome-wide association analyses (GWAS). Furthermore, GO and KEGG analyses revealed that the 10 candidate genes (MHC II, p38 MAPK, AMPK, SGK1, FOXO3, FOXO6, S1PR1, IL7R, RBL2, and GADD45) were related to intestinal immune network for IgA production pathway and FoxO signaling pathway. The acquisition of candidate genes related to resistance will help to explore the molecular mechanism of resistance to LMBV in largemouth bass. The potential polymorphic markers identified in this study are important molecular markers for disease resistance breeding in largemouth bass.

GADD45 (growth arrest and DNA damage inducible 45) is a crucial signaling regulator in cells and plays an important role in various biological processes, including cellular stress response, cell cycle control, DNA damage repair, apoptosis, and tumor suppression. Our previous studies identified GADD45b as a candidate gene associated with resistance to largemouth bass ranavirus (LMBV) infection in largemouth bass (Micropterus salmoides). In the present study, the upstream intergenic polymorphisms of GADD45b were investigated to explore their association with resistance/susceptibility to LMBV. We employed the kompetitive allele specific PCR (KASP) assay to genotype 118 resistant individuals and 122 susceptible individuals following LMBV infection. The results revealed that SNP38943374 C>A and SNP38943495 G>A were significantly associated with LMBV resistance/susceptibility (p < 0.01). Individuals with the CC genotype of SNP38943374 and the GG genotype of SNP38943495 were more prevalent in resistant groups and have advantages in survival time after LMBV infection. Linkage disequilibrium analysis indicated strong linkage among these two loci. The distinct dynamic expression patterns of GADD45b in different genotypes following LMBV infection suggest its functional role in viral infection. Additionally, dynamic expression levels of immune-related genes (IFN-γ, TNF-α, and IL-10) also varied among different genotypes. These results demonstrated that the two SNPs in GADD45b could be used as candidate markers for further investigation of selective breeding of resistant largemouth bass to LMBV.

Largemouth bass virus (LMBV) has caused severe impacts on the largemouth bass aquaculture industry in China. Breeding new virus-resistant strains is a fundamental strategy to address the LMBV challenge. In this study, a candidate LMBV-resistant population of largemouth bass was developed using the \"Youlu No. 3\" as the base population. Through three consecutive generations of selective breeding, a new LMBV-resistant strain was successfully established. To evaluate the LMBV resistance of different breeding generations, four key indicators, including survival rate post-LMBV infection, LMBV viral load, the expression of immune-related genes (GADD45b, FOXO3, TNF-α, IFN-γ, and IL-10), and antioxidant enzyme activities (GSH and AKP), were analyzed in this study. Notably, the F3 generation exhibited significantly lower viral loads in liver tissues after LMBV infection compared to the F1, F2, and control groups. Furthermore, the F3 generation showed a markedly higher survival rate at 14 days post-challenge, indicating enhanced disease resistance. Additionally, both the F2 and F3 generations had increased expression levels of the immune-related genes and elevated activities of antioxidant-related enzymes. These results collectively indicate that the F3 generation possesses stronger LMBV resistance than the F2, F1, and control groups. This study provides effective strategy for addressing LMBV disease in largemouth bass at the source and is of great significance for promoting the healthy and sustainable development of the largemouth bass aquaculture industry.

Tripartite Motif-Containing 44 (TRIM44) is responsible for cancers, neurodegenerative diseases, and viral infections. However, the role of Siniperca chuatsi TRIM44 (scTRIM44) during viral infection remains unclear. In the present study, we analyzed the molecular characteristics of scTRIM44 and its role in infectious spleen and kidney necrosis virus (ISKNV), largemouth bass virus (LMBV), and Siniperca chuatsi rhabdovirus (SCRV) infection. ScTRIM44 contained one B-box domain (B, 166–207 aa) and a coiled-coil domain (CC, 279–309 aa), but lacked the canonical RING domain of E3 ubiquitin ligases. The scTRIM44 mRNA was expressed relatively high in immune-related tissues. The mRNA expression of scTRIM44 significantly decreased in vivo and vitro post-ISKNV and -LMBV infection. However, the expression of scTRIM44 mRNA showed significant up-regulation post-SCRV infection. ScTRIM44 positively regulated SCRV infection in CPB cells, but copies of ISKNV and LMBV showed no significant alteration in over-expressed or knocked-down scTRIM44 cells. Moreover, scTRIM44 positively regulated RIG-I- and MDA5-mediated interferon molecule signaling. These data suggested that scTRIM44 promoted SCRV infection by positively regulating RIG-I- and MDA5-mediated interferon molecule signaling, but didn’t regulate ISKNV and LMBV infection. This research provided a comprehensive insight into the antiviral activity of scTRIM44.

Largemouth bass virus (LMBV) poses a significant threat to largemouth bass farming, leading to substantial economic losses. In December 2022, massive largemouth bass juveniles died at a fish farm in the city of Xinxiang, China. Through a series of experiments, we conclusively identified LMBV as the causative pathogen. The affected fish displayed anorexia, lethargy, and hemorrhage at the pectoral and caudal fin base. No parasites or pathogenic bacteria were detected on the body surface or gills, or isolated from the diseased fish. Severe hemorrhage, lymphocyte infiltration, and extensive necrosis were observed in the liver, spleen, intestine, and stomach of the moribund fish. The tissue homogenate from the diseased fish induced epithelioma papulosum cyprini cells (EPC) cell death, while no such effects were observed in grouper spleen (GS) cells. Sequence similarity analysis of the major capsid protein (MCP) indicated the virus shared 100% similarity with the LMBV-FS2021 strain, placing it within the Ranavirus genus. Transmission electron microscopy (TEM) observations revealed plenty of hexagonal virions accumulated in the cytoplasm of infected EPC cells. Artificial infection demonstrated that LMBV-XX01 was highly fatal to Micropterus salmoides juveniles, with an LD 50 of 10 3.081 TCID 50 /fish. RT-qPCR detection confirmed that LMBV appeared in all sampled tissues of the challenged largemouth bass, with significantly higher viral loads detected in the liver and heart compared to other tissues. Additionally, we successfully obtained a highly purified recombinant MCP of LMBV and developed two strains of monoclonal antibodies targeting MCP of LMBV-XX01. Overall, our findings provide valuable materials and insights for the design of prevention strategies and the development of detection methods for LMBV.

Background: Vaccinations are still the most effective means of preventing and controlling fish viral diseases, and cells are an important substrate for the production of a viral vaccine. Therefore, the rapid-stable growth and virus sensitivity of cells are urgently needed. Methods: Chinese perch brain 100th passage (CPB p100) were acclimated in a low serum with 5% FBS L-15 for 50 passages, then transferred to 8% FBS L-15 for 150 passages. Additionally, the morphology and cell type of CPB 300th passage (CPB p300) cells were identified. We analyzed the transfection efficiency and virus sensitivity of CPB p300 cells, and then optimized the conditions of ISKNV, SCRV, and LMBV multiplication in CPB cells. Results: CPB p300 cells were more homogeneous, and the spread diameter (20–30) µm in CPB p300 cells became the dominant population. The doubling time of CPB p300 was 1.5 times shorter than that of CPB p100.However, multiplication rate of CPB p300 was 1.37 times higher than CPB p100. CPB p300 cells were susceptible to ISKNV, SCRV, and LMBV, and the optimal conditions of ISKNV, SCRV, and LMBV multiplication were simultaneous incubation, 0.6 × 105 cells/cm2 and MOI = 0.1; infection at 48 h, 0.8 × 105 cells/cm2 and MOI = 0.01; simultaneous incubation, 0.7 × 105 cells/cm2 and MOI = 0.05, respectively. The time and economic costs of ISKNV, SCRV, and LMBV multiplication in CPB p300 cells were significantly reduced. Conclusions: The acquisition of CPB p300 cells laid a good material foundation for the production of ISKNV, SCRV, and LMBV vaccines.

The largemouth bass is a freshwater aquacultured fish species of great economic importance in China. With the rapid development of aquaculture industry and the increase in the aquaculture density of the fish, various infectious pathogens, including parasites, bacteria, and viruses, have been widely spread, which have caused huge losses to the aquaculture industry. Among them, largemouth bass iridovirus (LMBV) is one of the most harmful pathogens. In the present study, a virus strain named LMBV-GDSD was isolated from cultured largemouth bass and was successfully proliferated in FHM and EPC cells, with numerous viral particles observed in the infected cells under transmission electron microscopy analysis. The annotated complete genome of LMBV-GDSD was 99,285 bp and contained 102 ORFs. Based on genomic sequence alignment and phylogenetic analysis, the identified LMBV-GDSD belonged to the genus Ranavirus of Iridoviridae and was pathogenic to largemouth bass under regression infection experiments. In addition, the infection of LMBV-GDSD in largemouth bass could significantly up-regulate the expression of antiviral immune-related genes such as IRF3, IRF7, and Mx. It is thus providing valuable genetic data for a deeper understanding of the pathogenic mechanism of iridovirus in largemouth bass.

This chapter contains sections titled: Epizootic hematopoietic necrosis Lymphocystis Viral erythrocytic necrosis Viral nervous necrosis Red Sea bream iridoviral disease Other fish viruses Fish tumor viruses Management of other fish viruses References