Tilapia lake virus causes mitochondrial damage: a proposed mechanism that leads to extensive death in fish cells.

Background: Tilapia lake virus (TiLV), also known as Tilapinevirus tilapiae, poses a significant threat to tilapia aquaculture, causing extensive mortality and economic losses. Understanding the mechanisms and pathogenesis of TiLV is crucial to mitigate its impact on this valuable fish species. Methodology: In this study, we utilized transmission electron microscopy to investigate the ultrastructural changes in E-11 cells following TiLV infection. We also examined the presence of TiLV particles within the cells. Cellular viability and mitochondrial functions were assessed using MTT and ATP measurement assays and mitochondrial probes including JC-1 staining and MitoTracker™ Red. Results: Our findings provide novel evidence demonstrating that TiLV causes cytotoxicity through the destruction of mitochondria. Transmission electron micrographs showed that TiLV particles were present in the cytoplasm of E-11 cells as early as 1 h after infection. Progressive swelling of mitochondria and ultrastructural damage to the cells were observed at 1, 3 and 6 days post-infection. Furthermore, losses of mitochondrial mass and membrane potential (MMP) were detected at 1 day after TiLV inoculation, as determined by mitochondrial probes. The results of the MTT assay also supported the hypothesis that the cell deaths in E-11 cells during TiLV infection may be caused by the disruption of mitochondrial structure and function. Conclusions: Our study reveals the significant role of mitochondrial disruption in contributing to cellular death during the early stages of TiLV infection. These findings advance the understanding of TiLV pathogenesis and further enhance our knowledge of viral diseases in fish.

Assessment of Tilapia (Oreochromis spp.) Welfare in the Semi-Intensive and Intensive Culture Systems in Thailand.

Welfare assessments have risen to prominence in the aquaculture industry, with increasing awareness of their significance among stakeholders in Thailand. In this study, we conducted a welfare assessment of tilapia (Oreochromis spp.) farms in Thailand, focusing on health, environmental, behavioural, and nutritional indicators. Comparing semi-intensive (earthen ponds) and intensive farming practices (cage culture), we found significant differences in the overall health score, particularly at farm F due to a disease outbreak (Kruskal-Wallis, p = 0.01). Skin and fin scores varied across farms, indicating their potential as indicators of tilapia health. Environmental assessments revealed differences in transparency between the two culturing systems (Mann-Whitney, p = 0.02). During the harvesting process, tilapia behaviours indicated poor welfare across all farms. However, no statistically significant difference in overall welfare scores was found between the two culturing systems. Correlations were observed between nutritional, environmental, and health indicators, with negative correlations between fish density and water transparency (r = -0.87, p = 0.02), presence of inhabitants (r = -0.78, p = 0.04), feeding behaviours (r = -0.78, p = 0.04), and swimming behaviours during capture (r = -0.98, p = 0.001). These findings provide valuable insights to enhance tilapia-farming practices and welfare in Thailand.

The Modulation of Immune Responses in Tilapinevirus tilapiae-Infected Fish Cells through MAPK/ERK Signalling.

Tilapia lake virus (TiLV) is a novel RNA virus that has been causing substantial economic losses across the global tilapia industry. Despite extensive research on potential vaccines and disease control methods, the understanding of this viral infection and the associated host cell responses remains incomplete. In this study, the involvement of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway in the early stages of TiLV infection was investigated. The results showed a distinct pattern of ERK phosphorylation (p-ERK) upon TiLV infection in two fish cell lines, E-11 and TiB. Specifically, the p-ERK levels in the TiB cells decreased substantially, while the p-ERK levels in the E-11 cells remained constant. Interestingly, a large number of cytopathic effects were observed in the infected E-11 cells but none in the infected TiB cells. Furthermore, when p-ERK was suppressed using the inhibitor PD0325901, a significant reduction in the TiLV load and decrease in the mx and rsad2 gene expression levels were observed in the TiB cells in days 1-7 following infection. These findings highlight the role of the MAPK/ERK signalling pathway and provide new insights into the cellular mechanisms during TiLV infection that could be useful in developing new strategies to control this virus.

Tilapia lake virus (TiLV) causes severe anaemia and systemic disease in tilapia.

Tilapia lake virus disease (TiLVD) is an emerging disease in tilapia that is associated with mass mortality affecting global tilapia aquaculture. In this study, red hybrid tilapias (Oreochromis spp.) were experimentally infected by intracoelomic injection with Tilapia lake virus (TiLV) to gain a better understanding of the clinicopathological changes during infection. Pale bodies and gill were observed in infected fish after 7 days of post-challenge (dpc) associated with severe anaemia. Further haematological analysis in TiLV-infected fish revealed decreased levels of haemoglobin and haematocrit at 3 dpc. Common pathological findings included pale and friable liver, pale intestine with catarrhal content, and dark and shrunken spleen in TiLV-infected fish at 7 dpc and 14 dpc. Histologically, reduced numbers of red blood cells and accumulation of melano-macrophage centre in the spleen were found in infected fish at 3 dpc, and severe lesions were more commonly observed at 7 and 14 dpc. Lymphocyte infiltration, syncytial cell formation and multifocal necrotic hepatitis were the prominent pathological findings in the liver of infected fish. The severity of pathological changes was associated with TiLV-infection with higher viral loads and with the expression pattern of pro-inflammatory cytokines and antiviral genes, including interferon regulatory factor 1 (irf1), interleukin (il-8), radical s-adenosyl methionine domain containing 2 (rsad2) and mx. Our study provides a comprehensive analysis of the haematological profile and pathological changes in tilapia during TiLV infection. Overall, lesions present in various organs, together with alteration of host immune response in TiLV-infected fish, indicate the systemic infection of this virus. The knowledge gained from this study improves our understanding of how TiLV causes pathological and haematological changes in tilapia.

Antiviral Activity of Ribavirin against Tilapia tilapinevirus in Fish Cells.

The outbreak of the novel Tilapia tilapinevirus or Tilapia lake virus (TiLV) is having a severe economic impact on global tilapia aquaculture. Effective treatments and vaccines for TiLV are lacking. In this study, we demonstrated the antiviral activity of ribavirin against TiLV in E-11 cells. Our findings revealed that at concentrations above 100 µg/mL, ribavirin efficiently attenuates the cytopathic effect of the TiLV infection in fish cells. When administered in a dose-dependent manner, ribavirin significantly improved cell survival compared to the untreated control cells. Further investigation revealed that the cells exposed to ribavirin and TiLV had a lower viral load (p < 0.05) than the untreated cells. However, at concentrations above 1000 µg/mL, ribavirin led to cell toxicity. Taken together, our results demonstrate the efficacy of this antiviral drug against TiLV and could be a useful tool for future research on the pathogenesis and replication mechanism of TiLV as well as other piscine viruses.

Novel Approach to Assess Cardiac Function Using Systolic Performance and Myocardial Performance Indices From Simultaneous Electrocardiography and Phonocardiography Recordings in Dogs With Various Stages of Myxomatous Mitral Valve Disease.

Background and Objective: Myxomatous mitral valve disease (MMVD) progression entails changes in the structural and functional properties of the heart affecting cardiac timings and intervals within the cardiac cycle. Conventionally, echocardiography is used to determine the cardiac time intervals (CTIs) including systolic and myocardial performance indices (SPI and MPI) in evaluating cardiac function. Alternatively, these CTIs can also be measured using simultaneous recordings of electrocardiography (ECG) and phonocardiography (PCG), but their values in different MMVD stages remain to be established. This study aimed to establish and prove the use of derived SPI and MPI from a dedicated device as a novel approach to assess cardiac function in different stages of MMVD dogs. Materials and Methods: A prospective study in 52 dogs with different MMVD stages measured the CTIs using a novel device. These were compared and correlated with standard echocardiographic parameters. The predictive value of SPI and three new proposed formulas to estimate MPI (i.e., F1, F2, and F3) in association with asymptomatic from symptomatic MMVD dogs were investigated. Results: Our findings revealed that CTI parameters measured from a novel device including QS1, QS2, S1S2, MPI-F1, and MPI-F2 were altered at different stages of MMVD. The SPI and all proposed MPI formulas were comparable with the systolic time interval and Tei index from echocardiography. In addition, the SPI, MPI-F1, and MPI-F2 were significantly correlated with the Tei index. However, the SPI was not able to differentiate the various stages of MMVD. Conversely, only the MPI-F1 (i.e., (QS1 + S2)/S1S2) demonstrated good predictive accuracy when compared between asymptomatic and symptomatic MMVD dogs similar to the Tei index. Moreover, this formula was able to differentiate stages B1 and C with remarkable predictive accuracy, higher sensitivity, and high specificity when compared with the Tei index. Conclusion: We have successfully described the CTI parameters in different MMVD stages using simultaneous ECG and PCG recordings in dogs. Furthermore, we have proven that the concept of using the newly proposed parameters from a novel device is equivalent to the Tei index. Thus, we established a novel approach to evaluate cardiac function and its supportive use in the diagnosis of MMVD patients.

Negative inotropic mechanisms of ß-cardiotoxin in cardiomyocytes by depression of myofilament ATPase activity without activation of the classical ß-adrenergic pathway.

Beta-cardiotoxin (ß-CTX) from the king cobra venom (Ophiophagus hannah) was previously proposed as a novel ß-adrenergic blocker. However, the involvement of ß-adrenergic signaling by this compound has never been elucidated. The objectives of this study were to investigate the underlying mechanisms of ß-CTX as a ß-blocker and its association with the ß-adrenergic pathway. The effects of ß-CTX on isolated cardiac myocyte functions, calcium homeostasis, the phosphorylation level of targeted proteins, and the myofibrillar ATPase activity were studied. Healthy Sprague Dawley rats were used for cardiomyocytes isolation. Like propranolol, ß-CTX attenuated the cardiomyocyte inotropy and calcium transient alterations as induced by isoproterenol stimulation. In contrast, these effects were not observed in forskolin-treated cells. Interestingly, cardiomyocytes treated with ß-CTX showed no changes in phosphorylation level at any PKA-targeted sites in the myofilaments as demonstrated in Western blot analysis. The skinned fibers study revealed no change in myofilament kinetics by ß-CTX. However, this protein exhibited the direct inhibition of myofibrillar ATPase activity with calcium de-sensitization of the enzyme. In summary, the negative inotropic mechanism of ß-CTX was discovered. ß-CTX exhibits an atypical ß-blocker mechanism. These properties of ß-CTX may benefit in developing a novel agent aid to treat hypertrophic cardiomyopathy.

Suppression of cardiomyocyte functions by ß-CTX isolated from the Thai king cobra (Ophiophagus hannah) venom via an alternative method.

BACKGROUND: Beta-cardiotoxin (ß-CTX), the three-finger toxin isolated from king cobra (Ophiophagus hannah) venom, possesses ß-blocker activity as indicated by its negative chronotropy and its binding property to both ß-1 and ß-2 adrenergic receptors and has been proposed as a novel ß-blocker candidate. Previously, ß-CTX was isolated and purified by FPLC. Here, we present an alternative method to purify this toxin. In addition, we tested its cytotoxicity against different mammalian muscle cell types and determined the impact on cardiac function in isolated cardiac myocyte so as to provide insights into the pharmacological action of this protein. METHODS: ß-CTX was isolated from the crude venom of the Thai king cobra using reverse-phased and cation exchange HPLC. In vitro cellular viability MTT assays were performed on mouse myoblast (C2C12), rat smooth muscle (A7r5), and rat cardiac myoblast (H9c2) cells. Cell shortening and calcium transient dynamics were recorded on isolated rat cardiac myocytes over a range of ß-CTX concentration. RESULTS: Purified ß-CTX was recovered from crude venom (0.53% w/w). MTT assays revealed 50% cytotoxicity on A7r5 cells at 9.41 ± 1.14 µM (n = 3), but no cytotoxicity on C2C12 and H9c2 cells up to 114.09 µM. ß-CTX suppressed the extend of rat cardiac cell shortening in a dose-dependent manner; the half-maximal inhibition concentration was 95.97 ± 50.10 nM (n = 3). In addition, the rates of cell shortening and re-lengthening were decreased in ß-CTX treated myocytes concomitant with a prolongation of the intracellular calcium transient decay, indicating depression of cardiac contractility secondary to altered cardiac calcium homeostasis. CONCLUSION: We present an alternative purification method for ß-CTX from king cobra venom. We reveal cytotoxicity towards smooth muscle and depression of cardiac contractility by this protein. These data are useful to aid future development of pharmacological agents derived from ß-CTX.

Suppression of cardiomyocyte functions by ß-CTX isolated from the Thai king cobra (Ophiophagus hannah) venom via an alternative method

Beta-cardiotoxin (ß-CTX), the three-finger toxin isolated from king cobra (Ophiophagus hannah) venom, possesses ß-blocker activity as indicated by its negative chronotropy and its binding property to both ß-1 and ß-2 adrenergic receptors and has been proposed as a novel ß-blocker candidate. Previously, ß-CTX was isolated and purified by FPLC. Here, we present an alternative method to purify this toxin. In addition, we tested its cytotoxicity against different mammalian muscle cell types and determined the impact on cardiac function in isolated cardiac myocyte so as to provide insights into the pharmacological action of this protein. Methods: ß-CTX was isolated from the crude venom of the Thai king cobra using reverse-phased and cation exchange HPLC. In vitro cellular viability MTT assays were performed on mouse myoblast (C2C12), rat smooth muscle (A7r5), and rat cardiac myoblast (H9c2) cells. Cell shortening and calcium transient dynamics were recorded on isolated rat cardiac myocytes over a range of ß-CTX concentration. Results: Purified ß-CTX was recovered from crude venom (0.53% w/w). MTT assays revealed 50% cytotoxicity on A7r5 cells at 9.41 ± 1.14 µM (n = 3), but no cytotoxicity on C2C12 and H9c2 cells up to 114.09 µM. ß-CTX suppressed the extend of rat cardiac cell shortening in a dose-dependent manner; the half-maximal inhibition concentration was 95.97 ± 50.10 nM (n = 3). In addition, the rates of cell shortening and re-lengthening were decreased in ß-CTX treated myocytes concomitant with a prolongation of the intracellular calcium transient decay, indicating depression of cardiac contractility secondary to altered cardiac calcium homeostasis. Conclusion: We present an alternative purification method for ß-CTX from king cobra venom. We reveal cytotoxicity towards smooth muscle and depression of cardiac contractility by this protein. These data are useful to aid future development of pharmacological agents derived from ß-CTX.(AU)