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.

Widespread occurrence of Tilapia parvovirus in farmed Nile tilapia Oreochromis niloticus from India.

Tilapia parvovirus (TiPV) has been associated with heavy mortalities in tilapia as a single infection or in co-infection with Tilapia lake virus (TiLV). In this study, TiPV was detected in farmed Nile tilapia, Oreochromis niloticus, from two geographical regions of India, Maharashtra and Uttar Pradesh. TiPV-specific polymerase chain reaction (PCR) reported earlier was used in the screening. Tilapia collected from Maharashtra showed characteristic clinical signs, and TiPV was detected along with TiLV and/or Aeromonas spp. However, fish from Uttar Pradesh were apparently healthy and only TiPV could be detected in these samples. A high prevalence of TiPV was recorded from both the geographical locations, Maharashtra and Uttar Pradesh (59.6% and 95.0% respectively). The virus could be detected in tissues such as the spleen, liver, kidney, brain and mucus. The spleen appeared to be the best tissue for detecting TiPV in apparently healthy tilapia. The presence of TiPV was further confirmed through sequencing the PCR products, isolation of the virus in the cell line and electron microscopy. Sequences of the NS1 gene of the two TiPV isolates showed similarity to the earlier reported TiPV isolates. The virus could be successfully propagated in O. niloticus Liver (OnL) cell line, and cytopathic effect was observed as early as 3 days post-infection. Furthermore, the presence of non-enveloped icosahedral to round virus particles measuring about 26-35 nm could be demonstrated in the cytoplasm and nucleus of infected OnL cells in transmission electron microscopy. With this confirmation of the presence of the virus, India is the third country to report TiPV after China and Thailand. The detection of TiPV in co-infection cases with TiLV and in apparently healthy Nile tilapia suggests its wide distribution and potential synergistic effect in co-infection cases. Therefore, this emerging virus needs holistic attention to understand its virulence, host-specificity and epidemiological risk factors.

Susceptibilities of ten fish cell lines to infection with Tilapia lake virus.

Tilapia lake virus disease (TiLVD) caused by Tilapia lake virus (TiLV) is a great threat to the global tilapia culture industry. Effective prevention and control strategies have not been developed due to limited basic research of pathogenesis of TiLVD. Cell lines from different fish species have been found to be permissive to TiLV infection. In the current study, we comprehensively analyzed TiLV susceptibilities to 10 permanent growing fish cell lines. We found that the highest viral titers were generated onto TiB cells originated from the tilapia species Oreochromis mossambicus, MSF from the largemouth bass Micropterus salmoides, CAMK from the hybrid snakehead Channa argus × Channa maculata and SS derived from the perch species Siniperca chuatsi. Viral copy numbers from these four cell lines ranged from 4 × 107 copies/µL to 4.6 × 108 copies/µL. Confocal immunofluorescent microscopy also indicated that all 10 cell lines can support varying degrees of viral infection and replication. TiLV particles can be observed in cells from randomly selected three fish species using electron microscope. This study will assist in research and development of prevention and control of TiLVD.

Expression and purification of S5196-272 and S6200-317 proteins from Tilapia Lake Virus (TiLV) and their potential use as vaccines.

Tilapia Lake Virus Disease (TiLVD) is caused by Tilapia Lake Virus (TiLV), and it has a cumulative mortality rate of up to 90% in Nile tilapia (Oreochromis niloticus). TiLV is a negative enveloped single-stranded RNA virus with 10 genomic segments. Segment 5 (S5) and segment 6 (S6) were predicted to include a signaling peptide, suggesting that the encoded proteins of these two segments may exist as part of the virus envelope. Based on bioinformatic predictions, the S5 and S6 proteins in this study were produced, including S527-343, S527-172, S5196-272, S630-317, S630-190, and S6200-317. All proteins were tested for their expression in Escherichia coli. Only S5196-272 and S6200-317 were expressed as soluble and insoluble proteins, respectively. The soluble protein was purified using affinity chromatography, whereas the insoluble protein was solubilized using 6 M urea lysis buffer before purification. Both proteins were further purified using gel filtration chromatography, and the results showed a symmetric peak of both proteins suggested a high degree of uniformity in the conformation of these proteins. Antigenicity results indicated that these proteins were recognized by serum from TiLV-infected fish. The immunization tests revealed that serum antibodies levels in Nile tilapia produced by S5196-272 and S6200-317 were significantly increased (p-value < 0.05) at 7 days post-immunization (dpi) compared to antibody levels on Day 0 (D0). All the results combined suggested a potential vaccine candidate of S5 and S6 for TiLV protection in Nile tilapia.

An Age-Structured Model for Tilapia Lake Virus Transmission in Freshwater with Vertical and Horizontal Transmission.

This paper proposes a mathematical model for tilapia lake virus (TiLV) transmission in wild and farmed tilapias within freshwater. This model takes into account two routes of transmission: vertical and horizontal. This latter route integrates both the direct and indirect transmission. We define an explicit formula for the reproductive number [Formula: see text] and show by means of the Fatou's lemma that the disease-free equilibrium is globally asymptotically stable when [Formula: see text]. Furthermore, we find an explicit formula of the endemic equilibria and study its local stability as well as the uniform persistence of the disease when [Formula: see text]. Finally, a numerical scheme to solve the model is developed and some parameters of the model are estimated based on biological data. The numerical results illustrate the role of routes of transmission on the epidemic evolution.

Assessing the population transmission dynamics of tilapia lake virus in farmed tilapia.

A novel virus, tilapia lake virus (TiLV), has been identified as a key pathogen responsible for disease outbreak and mass mortality of farmed tilapia. We used a deterministic susceptible-infectious-mortality (SIM) model to derive key disease information appraised with published TiLV-induced cumulative mortality data. The relationship between tilapia mortality and TiLV exposure dosages was described by the Hill model. Furthermore, a disease control model was proposed to determine the status of controlled TiLV infection using a parsimonious control reproduction number (RC )-control line criterion. Results showed that the key disease determinants of transmission rate and basic reproduction number (R0 ) could be derived. The median R0 estimate was 2.59 in a cohabitation setting with 2.6 × 105  TCID50 fish-1 TiLV. The present RC -control model can be employed to determine whether TiLV containment is feasible in an outbreak farm by quantifying the current level of transmission. The SIM model can then be applied to predict what additional control is required to manage RC  < 1. We offer valuable tools for aquaculture engineers and public health scientists the mechanistic-based assessment that allows a more rigorous evaluation of different control strategies to reduce waterborne diseases in aquaculture farming systems.

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.

Reassortment and evolutionary dynamics of tilapia lake virus genomic segments.

The tilapia lake virus (TiLV), a highly infectious negative-sense single-stranded segmented RNA virus, has caused several outbreaks worldwide since its first report from Israel in 2014, and continues to pose a major threat to the global tilapia industry. Despite its economic importance, little is known about the underlying mechanisms in the genomic evolution of this highly infectious viral pathogen. Using phylogenomic approaches to the genome sequences of TiLV isolates from various geographic regions, we report on the pervasive role of reassortment, selection, and mutation in TiLV evolution. Our findings provided the evidence of genome-wide reassortment in this newly discovered RNA virus. The rate of non-synonymous (dN) to synonymous (dS) substitutions was less than one (dN/dS = 0.076 to 0.692), indicating that each genomic segment has been subjected to purifying selection. Concurrently, the rate of nucleotide substitution for each genomic segment was in the order of 1-3 × 10-3 nucleotide substitutions per site per year, which is comparable to the rate of other RNA viruses. Collectively, in line with the results of the previous studies, our results demonstrated that reassortment is the dominant force in the evolution and emergence of this highly infectious segmented RNA virus.

A mathematical model for tilapia lake virus transmission with waning immunity.

The goal of this paper is to investigate the influence of the waning immunity on the dynamics of Tilapia Lake Virus (TiLV) transmission in wild and farmed tilapia within freshwater. We formulate a model for which susceptible individuals can contract the disease in two ways: (i) direct mode caused by contact with infected individuals; (ii) indirect mode due to the presence of pathogenic agents in the water. We obtain an age-structured model which combines both age since infection and age since recovery. We derive an explicit formula for the reproductive number R0 and show that the disease-free equilibrium is locally asymptotically stable when, R0<1. We discuss on the form of the waning immunity parameter and show numerically that a Hopf bifurcation may occur for suitable immunity parameter values, which means that there is a periodic solution around the endemic equilibrium when, R0>1.

Tilapia lake virus disease: Phylogenetic analysis reveals that two distinct clades are circulating in Israel simultaneously.

Tilapia lake virus (TiLV) is an emerging viral disease that affects several tilapia species in different countries since 2014. In 2017-2018, 129 samples were collected from 14 tilapia farms in Israel. Ninety samples represented TiLV-suspected cases (TSC), and 39 were used as control samples (CS). RT-qPCR was performed on 89 and 39 duplicate brain and liver tissue samples from TSC samples and CS, respectively. TiLV was diagnosed in 37 (40.1%) of TSC, and two of the CS samples (5%) were also positive for TiLV. Additional validation RT-PCR was performed on positive samples, and amplified products were sequenced. Maximum-likelihood phylogenetic analysis of segment-3 of 25 selected sequences revealed two distinct clades: one virtually identical to sequences from India and the second closely related to isolates from Ecuador, Thailand, Egypt and Peru, apparently imported to Israel from Thailand. Thus, our results indicate that at least two distinct clades of TiLV are circulating in Israel simultaneously. As of today, the number of TiLV sequences available in free publicly accessible databases is limited. Nevertheless, our study provides new molecular epidemiology baseline for further epidemiological studies of TiLV.