Tilapia Lake Virus Infection Research Articles

Platycodin D: A promising anti tilapia lake virus natural compound from Platycodon grandiflorus

Tilapia lake virus (TiLV) poses a significant threat to the worldwide tilapia aquaculture sector, and there currently exists no efficient method of containment for this lethal pathogen. Given the widespread utilization of herbal medicines in the treatment of various aquatic animal diseases, it becomes imperative to evaluate and identify herbs that can be used for developing effective drugs against TiLV. We evaluated the efficacy of 20 herbs to inhibit TiLV proliferation in a TiLV infection model (Oreochromis niloticus) through two rounds of screening, with Platycodon grandiflorus extract showing the strongest inhibitory effect on TiLV replication (IC50 = 72.619 mg/L), survival of TiLV-infected tilapia was also improved in individual experiments. Platycodin D (PD), the principal bioactive compound found in Platycodon grandiflorus, demonstrated a dose-dependent reduction in viral load when used for treatment and enhanced the survival rate of tilapia infected with TiLV (42.3 %). The results of serum enzyme activity showed that PD treatment at a concentration of 5 mg/kg significantly enhance the non-specific parameters in tilapia (GSH, ACP, and AKP) (**p < 0.01). Additionally, immune-related gene analysis indicated that PD up-regulated IFN-I, CC2 and other genes expression while down-regulated the expression of pro-inflammatory factors (TNF-α, IL-1β) and TLR2 in the spleen and kidney of tilapia, implying that PD could induce IFN-I expression resulting in potential antiviral effects and inhibit some pro-inflammatory factors to alleviate oxidative stress and inflammatory responses in tilapia. The aforementioned findings suggest that Platycodon grandiflorus could potentially serve as a promising candidate for the development of drugs targeting TiLV. In particular, the bioactive component PD showed a strong anti-TiLV effect, which needs to be further explored.

Chromosome level genome assembly and transcriptome analysis of E11 cells infected by tilapia lake virus

The E11 cell line, derived from striped snakehead fish (Channa striata), possesses a distinctive feature: it is persistently infected with a C-type retrovirus. Notably, it exhibits high permissiveness to piscine nodavirus and the emerging tilapia lake virus (TiLV). Despite its popularity in TiLV research, the absence of genome assembly for the E11 cell line and Channa striata has constrained research on host-virus interactions. This study aimed to fill this gap by sequencing, assembling, and annotating the E11 cell line genome. Our efforts yielded a 600.5 Mb genome including 24 chromosomes with a BUSCO score of 98.8%. In addition, the complete proviral DNA sequence of snakehead retrovirus (SnRV) was identified in the E11 cell genome. Comparative genomic analysis between the E11 cell line and another snakehead species Channa argus revealed the loss of many immune-related gene families in the E11 cell genome, indicating a compromised immune response. We also conducted transcriptome analysis of mock- and TiLV-infected E11 cells, unveiling new perspectives on virus-virus and host-virus interactions. The TiLV infection suppressed the high expression of SnRV in E11 cells, and activated some other endogenous retroviruses. The protein-coding gene comparison revealed a pronounced up-regulation of genes involved in immune response, alongside a down-regulation of genes associated with specific metabolic processes.In summary, the genome assembly and annotation of the E11 cell line provide valuable resources to understand the SnRV and facilitate further studies on nodavirus and TiLV. The RNA-seq profiles shed light on the cellular mechanisms employed by fish cells in response to viral challenges, potentially guiding the development of therapeutic strategies against TiLV in aquaculture. This study also provides the first insights into the viral transcriptome profiles of endogenous SnRV and evading TiLV, enhancing our understanding of host-virus interactions in fish.

Tilapia lake virus infection disrupts the gut microbiota of red hybrid tilapia (Oreochromis spp.)

Viral infections have the potential to damage fish intestines and disrupt the composition and diversity of the bacterial populations. In this study, we investigated the gut microbiota profiles of 23 red hybrid tilapia (10 ± 3 g) infected with Tilapia lake virus (TiLV) via two different methods. For the intraperitoneal (IP)-injection, 50 μl of TiLV at a concentration of 105 TCID50/ml was administered into the fish's coelomic cavity. In the cohabitation challenge method, infection was achieved by injecting the virus into the coelomic cavity of the inducer fish and allow them to cohabited with cohabitation fish at a ratio of 1:3. The cohabitation infected fish had a cumulative mortality rate of 49%, while the IP-injection group had 81.5%, which was accompanied by clinical signs, severe histopathological changes in the intestines and high viral loads in the intestines over the infection period at 6, 12 and 21 days post challenge (dpc). Most importantly, an analysis of the bacterial populations based on the alpha and beta diversity patterns revealed distinct alterations in the intestines of the fish infected via IP injection and cohabitation and the control fish. Specifically, our findings highlighted a reduction in Cetobacterium (phylum Fusobacterium) and an increase in Brevinema (phylum Spirochaetes) in the infected fish at 6 and 12 dpc. Indeed, the elevation of Brevinema population is associated with a higher mortality rate within the IP-injection compared to the cohabitation group. Remarkably, the fish that survived TiLV infection at 21 dpc showed a higher abundance of Bacillus (phylum Firmicutes), which was in contrast to that at earlier time points (6 and 12 dpc). While Brevinema has been recognised as a potentially opportunistic pathogen in fish, different strains of Bacillus are commonly used as probiotics to promote fish health and enhance disease resistance. Our findings thus provide valuable insights into the distinct shifts occurring within fish gut microbiota during TiLV infection and suggest potential implications for improving gut health and resistance to viral infections in tilapia. Taken together, our study establishes that TiLV infection triggers significant changes in the diversity and composition of the intestinal microbiota in red hybrid tilapia.

Interferon regulatory factors inhibit TiLV replication by activating interferon-a3 in tilapia (Oreochromis niloticus)

Tilapia lake virus (TiLV) is an emerging virus that seriously threatens the tilapia industries worldwide. Interferon regulatory factors (IRFs), which are the crucial mediators regulating the response of interferon (IFN) to combat invading viruses, have not yet been reported in tilapia during TiLV infection. Here, six IRF (IRF1, IRF2, IRF4, IRF7, IRF8, and IRF9) homologs from tilapia were characterized and analyzed. These IRFs typically shared the conserved domains and phylogenetic relationship with IRF homologs of other species. Tissue distribution analysis showed that all six IRF genes were expressed in various tissues, with the highest expression in immune-related tissues. Furthermore, overexpression of IRFs in tilapia brain (TiB) cells significantly inhibited TiLV propagation, as evidenced by decreased viral segment 8 gene transcripts and copy numbers of viral segment 1. More importantly, all six IRF genes significantly enhanced the promoter activity of type I interferon-a3 (IFNa3) in TiB cells, suggesting that tilapia IRF genes serve as positive regulators in activating IFNa3. Surprisingly, the promoter activity of IFNa3 mediated by IRF genes was markedly inhibited post-TiLV infection, indicating that TiLV antagonized IRF-mediated IFN immune response. Taken together, six IRF genes of tilapia are highly conserved transcription factors that inhibit TiLV infection by activating the promoter of IFNa3, which is in turn restrained by TiLV. These findings broaden our knowledge about the functionality of IRF-mediated antiviral immunity in tilapia against TiLV infection and host-TiLV interaction, which lays a foundation for developing antiviral strategies in tilapia cultural industries.

Coxsackievirus and adenovirus receptor inhibits tilapia lake virus infection via binding to viral segment 8 and 10 encoded protein

The global aquaculture industry of tilapia (Oreochromis niloticus) has been significantly impacted by the emergence of tilapia lake virus (TiLV). However, effective prevention and control measures are still not available due to a lack of unclear pathogenesis of TiLV. Our previous transcriptome found that coxsackievirus and adenovirus receptor (CAR) was in response to TiLV infection in tilapia. To explore the potential function of OnCAR, the effect of OnCAR on TiLV proliferation was analyzed in this study. The OnCAR open reading frame (ORF) sequence of tilapia was 516 bp in length that encoded 171 amino acids with an Ig-like domain and transmembrane region. The OnCAR gene showed widespread expression in all investigated tissues, with the highest levels in the heart. Moreover, the OnCAR gene in the liver and muscle of tilapia exhibited dynamic expression levels upon TiLV challenge. Subcellular localization analysis indicated that OnCAR protein was mainly localized on the membrane of tilapia brain (TiB) cells. Importantly, the gene transcripts, genome copy number, S8-encoded protein, cytopathic effect, and internalization of TiLV were obviously decreased in the TiB cells overexpressed with OnCAR, indicating that OnCAR could inhibit TiLV replication. Mechanically, OnCAR could interact with viral S8 and S10-encoded protein. To the best of our knowledge, OnCAR is the first potential anti-TiLV cellular surface molecular receptor discovered for inhibiting TiLV infection. This finding is beneficial for better understanding the antiviral mechanism of tilapia and lays a foundation for establishing effective prevention and control strategies against tilapia lake virus disease (TiLVD).

Analisis Molekuler Tilapia Lake Virus (TiLV) di Sentra Budidaya Benih Ikan Gurami (Osphronemus goramy) Jawa Timur

Tilapia Lake Virus (TiLV) is a New Emerging Disease that has been defined and included in the WOAH disease list (WOAH, 2022). This virus can infect both freshwater and marine fish, causing high mortality in Ttilapia up to 90%. Reverse transcyptase method - Semi Nested Polymerase Chain Reaction (RT-snPCR) for detection of TiLV in Osphronemus goramy. TiLV was discovered for the first time in Thailand and was also found in Jogjakarta in 2021. Further research is needed to see the damage and genetic variation caused by TiLV in Osphronemus goramy. This study aims to study the genetic variability and homology of TiLV isolates in Osphronemus goramy as definitive hosts in East Java. The results showed clinical symptoms of lethargic fish, decreased appetite, abnormal behavior, reduced reflexes, sluggish movements, darker body color, congestion, thinning fins and exopthalmia. The PCR results showed that the Osphronemus goramy showing clinical symptoms of TiLV infection were all positive. The sequencing results were further identified using the BLAST program, showing the highest similarity of the genetic identity of the sample at 96.8% with TiLV from Israel (Genebank Accession Number NC 029927.1).

Isolation, in vitro, and in vivo pathogenicity test of Tilapia lake virus (TiLV) and development of a prognostic semi-quantitative lesion scoring system for differentiating clinical/subclinical infection in farmed tilapia (Oreochromis niloticus L.)

Tilapia lake virus (‘TiLV-MH-2022’) was recently recovered from the naturally infected farmed tilapia. Reverse transcription-polymerase chain reaction (RT-PCR) using segment 1 specific primers, followed by Sanger sequencing, confirmed the infection. The pairwise sequence homology of segment 1 showed its close relationship with the previous isolates. The virus was successfully detected from the mucus, which emphasised the possibility of non-invasive screening of tilapia on a large scale. The virus inoculum prepared from the infected tissues was tested for in vivo and in vitro pathogenicity. Around 100-140 nm-sized electron-dense virus particles were observed in the infected OnlL cells. Based on the onset of symptoms and lesions, all RT-PCR-positive fish were categorised into two groups, ‘clinical’ and ‘subclinical’. A lesion-scoring technique was developed for assessing the pathogenicity of the virus isolate. The external and internal gross lesions and histopathological alterations in the critical organs of the fish, such as the brain, kidney, gills, and liver, were assessed on a scale of 0 (no gross lesion) to 5 (most severe lesions). Overall lesion score was significantly high in the clinical and subclinical groups for gross and histopathology, respectively. This study is the first such attempt to standardise a semi-quantitative lesion scoring technique for TiLV infection, which establishes a clinical relevance and prognostic ability to distinguish between the apparent and inapparent infection.

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

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. 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. 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. 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.

Effects of light and circadian clock on the antiviral immune response in zebrafish.

The circadian clock mechanism, which is evolutionarily conserved across various organisms, plays a crucial role in synchronizing physiological responses to external conditions, primarily in response to light availability. By maintaining homeostasis of biological processes and behavior, the circadian clock serves as a key regulator. This biological mechanism also coordinates diurnal oscillations of the immune response during infections. However there is limited information available regarding the influence of circadian oscillation on immune regulation, especially in lower vertebrates like teleost fish. Therefore, the present study aimed to investigate the effects of light and the timing of infection induction on the antiviral immune response in zebrafish. To explore the relationship between the timing of infection and the response activated by viral pathogens, we used a zebrafish model infected with tilapia lake virus (TiLV). Our findings demonstrated that light availability significantly affects the antiviral immune response and the functioning of the molecular clock mechanism during TiLV infection. This is evident through alterations in the expression of major core clock genes and the regulation of TiLV replication and type I IFN pathway genes in the kidney of fish maintained under LD (light-dark) conditions compared to constant darkness (DD) conditions. Moreover, infection induced during the light phase of the LD cycle, in contrast to nocturnal infection, also exhibited similar effects on the expression of genes associated with the antiviral response. This study indicates a more effective mechanism of the zebrafish antiviral response during light exposure, which inherently involves modification of the expression of key components of the molecular circadian clock. It suggests that the zebrafish antiviral response to infection is regulated by both light and the circadian clock.

Establishment of a tilapia lake virus (TiLV)-susceptible cell line for anti-TiLV Chinese herbal compound screening

Tilapia lake virus (TiLV) has caused many deadly epidemics in wild and farmed tilapia, posing a great threat to the global tilapia-based industry. It is necessary and urgent to develop effective measures for TiLV control. Previously, we isolated a TiLV strain from naturally diseased tilapia, and its main target tissues are liver, spleen and brain. In the study, a TiLV-susceptive cell line is established based on the tissue tropism, and anti-TiLV activity of 11 Chinese herbal compounds (CHCs) is checked in vitro. A cell line from the brain of tilapia, designated as Nt-B, can be continuously subcultured for >100 passages and maintain a good proliferating state in L-15 media supplemented with 10% FBS at 28 °C. Following infection with TiLV, a series of cytopathic effects can be observed, including rounding, accumulation, vacuolation and detachment of cells. Enveloped viral particles can be observed in TiLV-infected cells, and TiLV-specific bands are identified by PCR analysis, confirming susceptibility of Nt-B cells to TiLV. For anti-TiLV activity evaluation, the selected CHCs show inhibition activity on TiLV-induced CPEs in different degrees except CHC-I. Two kinds of CHCs, CHC-II and CHC-X, have inhibition activity on TiLV-induced CPEs higher than 80%. Besides, the 90% inhibitory concentrations of CHC-II and CHC-X on TiLV are 51.12 ± 3.04 and 68.28 ± 6.37 mg/L, respectively. In summary, the newly established Nt-B cell line provides a useful tool for TiLV-related studies, moreover, CHC-II is a promising therapeutic agent against TiLV infection. Further studies will be required to reveal the active compounds and anti-TiLV mechanism of CHC-II.

Immune responses to Tilapia lake virus infection: what we know and what we don't know.

Tilapia lake virus (TiLV) is a novel contagious pathogen associated with a lethal disease affecting and decimating tilapia populations on several continents across the globe. Fish viral diseases, such as Tilapia lake virus disease (TiLVD), represent a serious threat to tilapia aquaculture. Therefore, a better understanding of the innate immune responses involved in establishing an antiviral state can help shed light on TiLV disease pathogenesis. Moreover, understanding the adaptive immune mechanisms involved in mounting protection against TiLV could greatly assist in the development of vaccination strategies aimed at controlling TiLVD. This review summarizes the current state of knowledge on the immune responses following TiLV infection. After describing the main pathological findings associated with TiLVD, both the innate and adaptive immune responses and mechanisms to TiLV infection are discussed, in both disease infection models and in vitro studies. In addition, our work, highlights research questions, knowledge gaps and research areas in the immunology of TiLV infection where further studies are needed to better understand how disease protection against TiLV is established.

A nested model with boosting and waning of immunity from Tilapia Lake Virus infection with distributed resistance to pathogens carrier-state.

This paper proposes and analyzes an immune-structured population model of tilapia subject to Tilapia Lake Virus (TiLV) disease. The model incorporates within-host dynamics, used to describe the interaction between the pathogen, the immune system and the waning of immunity. Individuals infected with a low dose acquire a low immunity level and those infected with a high dose acquire a high level of immunity. Since individuals' immune status plays an important role in the spread of infectious diseases at the population level, the within-host dynamics are connected to the between-host dynamics in the population. We define an explicit formula for the reproductive number [Formula: see text] and show that the disease-free equilibrium is locally asymptotically stable when [Formula: see text], while it is unstable when [Formula: see text]. Furthermore, we prove that an endemic equilibrium exists. We also study the influence of the initial distribution of host resistance on the spread of the disease, and find that hosts' initial resistance plays a crucial role in the disease dynamics. This suggests that the genetic selection aiming to improve hosts' initial resistance to TiLV could help fight the disease. The results also point out the crucial role played by the inoculum size. We find that the higher the initial inoculum size, the faster the dynamics of infection. Moreover, if the initial inoculum size is below a certain threshold, it may not result in an outbreak at the between-host level. Finally, the model shows that there is a strong negative correlation between heterogeneity and the probability of pathogen invasion.

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.

Inulin acetate as a novel recombinant subunit vaccine carrier for immunization of tilapia against tilapia lake virus

Tilapia lake virus (TiLV) is highly contagious and pathogenic to tilapia, causing enormous economic losses in tilapia industry. Up to now, there is no commercial vaccine is available for against TiLV infection. Therefore, a novel vaccine with high immune efficacy is urgently-needed. In this study, we examined the efficacy of subunit vaccine against TiLV through intramuscular injection using inulin acetate (InAc) nanoparticles encapsulation. The S2 protein (encoded by segment 2 gene of TiLV) was used as the antigen and the delivery system was named as InAc-S2. The toxicity analysis showed InAc-S2 had no significant influence on fishes growing and development. Meanwhile, we found InAc-S2 has a dual-release pattern (initial burst release and sustained release) in antigen release. Subsequently, tilapia was injected intramuscularly by different treatments (PBS, InAc, S2, and InAc-S2), and the immune protective effect induced by these treatments was analyzed. We found a powerful and long-lasting humoral or cellular immune response (serum antibody production, immune-related parameters and immune-related genes expression) can be induced in fishes by injected with InAc-S2 in comparison with those injected with S2 antigen alone. Moreover, a relative higher survival rate was observed in fish vaccinated with InAc-S2 at the dose of 4 μg protein/g of fish (with the relative percentage survival of 58.6%). Overall, this study for the first time demonstrated the potential of InAc as subunit vaccine carrier against TiLV infection and provide a reference to novel vaccine development in aquaculture.

Development of an indirect ELISA test for the detection of Tilapia lake virus (TiLV) in fish tissue and mucus samples

A serological test for screening of TiLV in Oreochromis niloticus would be useful for the epidemiological investigations. Using polyclonal antisera against TiLV (TiLV-Ab), an indirect enzyme-linked immune sorbent assay (iELISA) was developed for the detection of TiLV antigen in fish tissue and mucus. After a cutoff value was established and antigen and antibody concentrations were optimized, the iELISA's sensitivity and specificity were assessed. We found the ideal dilutions of TiLV-Ab as 1: 4000 and secondary antibody as 1:65,000. High analytical sensitivity and moderate specificity were displayed by the developed iELISA. The Positive and Negative Likelihood Ratio (LR+, LR-) were 1.75 and 0.29, respectively. The estimated Positive and Negative Predictive Values (PPV and NPV) of the test were 76.19% and 65.62%, respectively. The accuracy of the developed iELISA was estimated as 73.28%. An immunological survey was performed using the developed iELISA with samples from the field and 155/195 fishes tested positive, indicating a 79.48% TiLV antigen positives. Among the pooled organs and mucus tested, the highest positive rate of 92.3% (36/39) is observed in mucus compared to other tissues, and least positive rate is found in liver of 46% (18/39). The newly designed iELISA proved sensitive and may be helpful for extensive examinations of TiLV infections and monitoring disease status even from apparently healthy samples using a non-invasive technology by collecting mucus as sample for iELISA.

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.

Disease development in red hybrid tilapia following single and co-infection with tilapia lake virus and Streptococcus agalactiae

Tilapia lake virus (TiLV) has recently been reported to cause infection in cultured and wild tilapia globally, and co-infection between TiLV and other bacterial pathogens contributes to higher mortality and economic losses. Understanding the pathology and pathogenesis of the co-infection could help in controlling the outbreaks. This study determines and compares the severity of single and co-infection of TiLV and Streptococcus agalactiae in red hybrid tilapia. The tilapias were challenged intra-peritoneal with 106 TCID50/mL of TiLV and 103 CFU/mL of S. agalactiae either as single or as co-infection. Fish were divided into five experimental groups with four replicates (30 fish/replicate). Group 1 received a single TiLV intra-peritoneal infection at 106 TCID50/mL of TiLV, Group 2 received a single S. agalactiae intra-peritoneal infection at 103 CFU/mL of S. agalactiae, Group 3 was co-infected intra-peritoneal with TiLV and S. agalactiae, Group 4 was co-infected with S. agalactiae and TiLV, and Group 5 was the control unchallenged. Co-infection by S. agalactiae in Group 3 was done at 13-days post infection (dpi), while co-infection by TiLV in Group 4 was at 2-dpi. Following challenge, fish were monitored daily for 20 days, while samples of brain and liver were collected at 2-dpi intervals for viral and bacterial load analyses. Co-infected fish recorded higher cumulative mortalities (60% for TiLV-S. agalactiae and 73% for S. agalactiae-TiLV) compared to single infection (40% for TiLV and 20% for S. agalactiae). Both single and co-infected fish showed similar clinical signs and gross lesions, respective to the exposed pathogen. Notable histopathological changes included the presence of multinucleated syncytial giant cells and intracytoplasmic inclusion bodies following TiLV infection either in single or co-infection, but not in S. agalactiae single infection. The mean of histopathological score for brain and liver, and pathogen loads were significantly (p < 0.05) higher in the co-infected fish. This study describes the disease development following TiLV and S. agalactiae co-infection and concluded that co-infection of TiLV and S. agalactiae increases the mortality and enhances the disease severity in tilapia.

New emerging viral disease on Giant Gourami (Osphronemus goramy Lac.) in Java, Indonesia

This study investigates the role of pathogens in the disease outbreaks and mass mortality affecting giant gourami (Osphronemus goramy) populations from 2018 to 2020, leading to significant economic losses. Presumptive diagnoses, based on clinical symptoms and references, implicated two viruses: Tilapia Lake Virus (TiLV) and Infectious Spleen and Kidney Necrosis Virus (ISKNV). Samples from four Indonesian provinces (West Java, Central Java, East Java and Yogyakarta) were tested molecularly for these viruses. The results revealed widespread viral pathogen involvement, with ISKNV prevalence ranging from 20% to 100% and TiLV prevalence consistently at 100%. These findings strongly suggest that the outbreaks were primarily caused by TiLV and ISKNV infections, potentially with other co-infecting pathogens. Bacterial pathogens, notably Aeromonas spp., Mycobacterium spp., and Pseudomonas spp., may have exacerbated the disease. This research highlights the emergence of viral diseases as a significant threat to giant gourami populations in Java, Indonesia.

Mapping of Tilapia Lake Virus entry pathways with inhibitors reveals dependence on dynamin activity and cholesterol but not endosomal acidification.

Tilapia Lake Virus (TiLV) is an emerging virus lethal to tilapia, which threatens the global tilapia aquaculture with severe implications for food security. TiLV possesses similar features to orthomyxoviruses but is classified in the sole and the monotypic genus Tilapinevirus of the family Amnoonviridae. TiLV enveloped virions encapsidate a genome comprising ten segments of single-stranded, negative RNA. Remarkably, nine of TiLV's ten major proteins lack sequence homology to any known viral or cellular proteins. The mode of TiLV entry into tilapia cells is not known. Following the measurement of the entry window of TiLV (∼3h), we applied a panel of inhibitors of known regulators of endocytic functions to map the molecular requirements for TiLV entry. We identified productive entry by quantification of TiLV nucleoprotein expression and the generation of infectious particles. Inhibition of dynamin activity with dynasore or dynole, or depletion of cholesterol with methyl-β-cyclodextrin, strongly inhibited TiLV protein synthesis and infectious virion production. Moreover, inhibition of actin cytoskeleton polymerization with latrunculin A or microtubule polymerization with nocodazole within the entry window resulted in partial inhibition of TiLV infection. In contrast, inhibitors of endosomal acidification (NH4Cl, bafilomycin A1, or chloroquine), an inhibitor of clathrin-coated pit assembly (pitstop 2), and erlotinib-an inhibitor of the endocytic Cyclin G-associated kinase (GAK), did not affect TiLV entry. Altogether, these results suggest that TiLV enters via dynamin-mediated endocytosis in a cholesterol-, cytoskeleton-dependent manner, and clathrin-, pH-independent manner. Thus, despite being an orthomyxo-like virus, when compared to the prototypical orthomyxovirus (influenza A virus), TiLV shows a distinct set of requirements for entry into cells.

Identification and pathogenetic study of tilapia lake virus (TiLV) isolated from naturally diseased tilapia

As an emerging pathogen, tilapia lake virus (TiLV) has caused severe socio-economic impacts and remains a devastating factor in wild and farmed tilapia. Early diagnosis and timely reporting of TiLV are very important and necessary. In the study, a TiLV infection event in a tilapia farm is studied and reported. Naturally diseased tilapia with clinical signs such as anorexia, exophthalmia, skin abrasion and hemorrhage are collected. Multiple tissue lesions of liver, spleen and kidney are observed by histopathological analysis, such as syncytial formation, intracytoplasmic inclusion body and necrosis. No bacterial pathogens are identified from liver, spleen and kidney of the diseased tilapia, while all of them show TiLV-positive. E-11 cells are employed for TiLV isolation, and cytopathic effects are developed following incubation with supernatants of the homogenized tissues collected from the diseased tilapia. Electron micrographs of the infected cells and purified TiLV show that nearly round-shaped, enveloped viral particles (60–80 nm) can be identified. Segment 1 of the isolated TiLV has a high sequence similarity to other isolates, but phylogenetic analysis shows that it is placed in a unique cluster. Experimental infection with the isolated TiLV can cause high mortality (> 90%) in healthy Nile tilapia, with the similar clinical signs and tissue lesions to those found in naturally infected tilapia. Moreover, vacuolation and necrosis in brain, inclusion body and necrosis in heart, megalocytes and necrosis in intestine are observed in experimentally infected tilapia. Muscle, spleen, liver, intestine, brain, heart, kidney and gill of the re-infected tilapia show TiLV-positive, and liver, spleen and brain are the main target tissues. The data so far confirm a TiLV-associated disease outbreak in China, enriching the relevant information of TiLV.