Tilapia Lake Virus Research Articles

An RT-RPA-Cas12a platform for rapid and sensitive detection of tilapia lake virus

Tilapia lake virus (TiLV) or Tilapia tilapinevirus is a highly contagious pathogen that causes severe symptoms and massive mortalities in tilapia, affecting numerous tilapia-farming communities across the continents. Though TiLV has grown into a serious threat that could undermine the aquaculture-based economy and global food security, effective therapeutics and vaccines are not yet commercially available, rendering prompt detection and control the most practical strategy. Current TiLV diagnostic techniques, however, face a number of constraints that restrict their utility, including low throughput and high resource demands imposed upon users. To fill in this critical gap, we developed a novel CRISPR-Cas12a method coupled with reverse-transcriptase recombinase polymerase amplification (RT-RPA-Cas12a) for TiLV detection, using Segment 9 as the target. The platform could detect as few as 200 copies of RNA and produced no false positive results when tested with other fish pathogens. The assay could be carried out at a constant temperature between 37 and 42 °C in less than an hour, allowing naked-eye interpretation of the results through lateral flow and smartphone-based readouts. Taken together, our RT-RPA-Cas12a platform is accurate, streamlined, and user-friendly, and thus has the potential to be a valuable asset in combating the growing threat posed by TiLV.

Mannose functionalized biomimetic nanovaccine enhances immune responses against tilapia lake virus

Tilapia lake virus disease (TiLVD) caused by tilapia lake virus (TiLV) is a lethality truculence viral disease endangering the sustainable development of tilapia aquaculture. DNA vaccine present immense application potential in disease prevention and control, but the low gene transfection efficacy of naked DNA vaccine imposes restrictions on itself immune response that should have been induced. In this study, we constructed a biomimetic nanodelivery system (Cs-pS2@M-M) using the mannose modified erythrocyte membrane acted as a DNA vaccine carrier. The nanoparticle has a typical core-shell structure with a particle size of ~100 nm, and ensures sustained and efficient expression of plasmid DNA in muscle and spleen tissue. Intramuscular immunization of tilapia to evaluate the efficacy of the vaccine, the results showed that it has a high-effective induction of serum antibody production, enzyme activity, and immune-related gene expression compared to the same dose (10 μg/g) of non-mannosylated Cs-pS2@M nanoparticles and naked DNA vaccine. Notably, the TiLV challenge trial show that Cs-pS2@M-M provided 76.9% relative percentage survival (RPS), which was 26.9% higher than the 50.0% of the naked DNA vaccine (pS2), and not only that, it was 15.4% higher than that of Cs-pS2@M without mannose modification. These results showed that Cs-pS2@M-M biomimetic nanovaccine constructed in this study elicited robust immune responses to protect tilapia from TiLV challenge, and erythrocyte membrane-coated nanoparticles strategy show enormous promise potential as a vaccine delivery platform for aquatic animals.

Immunoinformatic Approaches to Identify Immune Epitopes and Design an Epitope-Based Subunit Vaccine against Emerging Tilapia Lake Virus (TiLV)

Tilapia tilapinevirus, known worldwide as tilapia lake virus (TiLV), is a single-stranded RNA virus that belongs to the Amnoonviridae family. The virus attacks the fish species’ external and internal organs, such as the eyes, brain, and liver. Syncytial cells develop in the liver cells of infected fish, which are characterized by widespread hepatocellular necrosis and karyolytic nuclei. It is a highly infectious virus that spreads both horizontally and vertically. Despite these devastating complications, there is still no cure or vaccine for the virus. Therefore, a vaccine based on epitopes developed using immunoinformatics methods was developed against TiLV in fish. The putative polymerase basic 1 (PB1) gene was used to identify immunodominant T- and B-cell epitopes. Three probable epitopes were used to design the vaccine: CTL, HTL, and LBL. Testing of the final vaccine revealed that it was antigenic, non-allergenic, and has improved solubility. Molecular dynamics simulation revealed significant structural compactness and binding stability. Furthermore, the computer-generated immunological simulation indicated that immunization might stimulate real-life immune responses following injection. Overall, the findings of the study imply that the designed epitope vaccine might be a good option for prophylaxis for TiLV.

Tilapia Lake Virus (TiLV) from Vietnam is genetically distantly related to TiLV strains from other countries.

Tilapia Lake Virus (TiLV) is reported as a threat to tilapia aquaculture in 16 countries on four continents with outbreaks causing up to 90% mortality. This research is one of the first studies on TiLVs from Vietnam. We propagated successfully one TiLV isolate HB196-VN-2020 from a diseased tilapia sample using an E-11 cell line and evaluated its virulence in two different weights of red hybrid tilapia and three serial 10-fold diluted viral titers. Smaller fish (4.5 ± 1.98 g) were proved to be more susceptible to TiLV infection at the viral titre of 9.1 × 105 TCID50 fish-1 than larger fish (20.8 ± 7.5g) with the mortalities of 92.5% and 12.5%, respectively. Reassortant detection analysis revealed seven potential reassortment events among 23 TiLV genomes, indicating the mixed infection of multiple TiLV isolates at the farms and the fish movement among different regions. Seven maximum likelihood phylogenetic trees based on the individual segments or the concatenated coding regions of some segments showed the genetically distant relationship of the Southern Vietnamese isolate RIA2-VN-2019 with the 21 reference isolates, and suggest the different origins of two Vietnamese TiLV isolates (RIA2-VN-2019 and HB196-VN-2020). However, additional sequences from various sampling locations and times are required to better understand the impacts of genetic diversity and reassortments on the evolution, migration and natural selection of TiLVs in Vietnam and other countries.

Efficiency, sensitivity and specificity of a quantitative real-time PCR assay for Tilapia Lake virus (TiLV)

Tilapia lake virus (TiLV) is an emerging viral pathogen of tilapiines worldwide in wild and farmed tilapia. TiLV is an orthomyxo-like, negative sense segmented RNA virus, belonging to genus Tilapinevirus, family Amnoonviridae. Here we developed a quantitative real-time PCR (qRT-PCR) assay testing primer sets targeting the 10 segments of TiLV. Sensitivity, specificity, efficiency and reproducibility of these assays were examined. Detection sensitivity was equivalent to 2 TCID50/ml when tested on supernatants from cell culture-grown TiLV. Specificity tests showed that all primer sets amplified their respective TiLV segments, and standard curves showed linear correlation of R2 > 0.998 and amplification efficiencies between 93 % and 98 %. Intra- and inter-assay coefficients of variation (CV %) were in the range of 0.0 %− 2.6 % and 0.0 %− 5.9 %, respectively. Sensitivity tests showed that primer sets targeting segments 1, 2, 3 and 4 had the highest detection sensitivities (100.301 TCID50/ml). The qRT-PCR used for detection of viral genome in TiLV infected organs gave virus titers equivalent to 3.80 log10, 3.94 log10 and 3.52 log10 TCID50/ml for brain, kidney and liver tissues, respectively as calculated on the basis of Ct values. These findings suggest that primer optimization for qPCR should not only focus on attaining high amplification efficiency but also sensitivity comparison of primer sets targeting different viral segments in order to develop a method with the highest sensitivity.

A high efficacy DNA vaccine against Tilapia lake virus in Nile tilapia (Oreochromis niloticus)

Tilapia lake virus (TiLV) is the main tilapia-infecting virus worldwide, causing serious economic losses to the tilapia aquaculture. However, there is no vaccine for this viral disease. Here, TiLV ORF10 (TiLV-ORF10) encoding a protein with abundant epitopes was cloned into the eukaryotic expression vector pcDNA3.1 as a vaccine candidate, and then used to evaluate the protective effects against TiLV in Nile tilapia (Oreochromis niloticus). RT-PCR and Western blot analyses confirmed pcDNA3.1–ORF10 expression in tilapia. The transcription levels of immunity-related genes such as immunoglobulin M, Toll-like receptor 2, myeloid differentiation factor 88, interleukin 8, tumor necrosis factor alpha, gamma-IFN, and nuclear factor κB were significantly upregulated in the spleen, liver, and kidney of the vaccinated tilapias (P < 0.05). TiLV challenge experiments showed that relative percent survival (RPS) was significantly enhanced in fish vaccinated with the DNA vaccine. Moreover, the RPS was significantly higher in fish vaccinated with a high dose of the DNA vaccine (85.72% RPS at a dosage of 45 μg DNA vaccine plasmid). Meanwhile, vaccination with pcDNA3.1–ORF10 significantly reduced virus replication, as evidenced by the lower amount of virus in the spleen, liver, and kidney of vaccinated tilapia than tilapias vaccinated with the empty pcDNA3.1 plasmid. Thus, pcDNA3.1–ORF10 could induce protective immunity in tilapia and may be a potential vaccine candidate for controlling disease caused by TiLV.

Establishment and characterization of a continuous cell line from caudal fin of Labeo calbasu (Hamilton, 1822).

Labeo calbasu is an important food fish and candidate species for diversification of carp aquaculture. In the present study, we have established a continuous cell line, designated as L. calbasu fin(LCF), from caudal fin of L. calbasu using explant method. The cell line has been subcultured for over 73 passages and the LCF cells show optimal growth in Leibovitz's L-15 medium supplemented with 20% fetal bovine serum at a temperature of 28°C. In karyotype analysis, the modal chromosome number of LCF cells at 35th passage was found to be 50. The amplification and sequencing of partial fragments of mitochondrial genes, namely 16S rRNA and COI from LCF cells confirmed the origin of cell line from L. calbasu. The LCF cells could be successfully transfected with GFP reporter gene, indicating suitability of these cells for expression of foreign genes. Further, following inoculation with supernatant from Tilapia lake virus (TiLV) infected cell line, no cytopathic effects were observed in the LCF cells and cell pellet was negative for TiLV in RT-PCR, indicating that LCF cells were not susceptible to TiLV. The developed cell line has been submitted to National Repository of Fish Cell Lines being maintained at ICAR-National Bureau of Fish Genetic Resources, Lucknow (accession no. NRFC063). The newly developed LCF cell line would be helpful in investigating diseases affecting this candidate species particularly the ones suspected to be of viral etiology, and for cytotoxicity and transgenic studies.

Assessing the effect of probiotics on tilapia lake virus-infected tilapia: Transmission and immune response.

Probiotics have been used to alleviate disease transmission in aquaculture. However, there are limited studies on probiotic use in modulating tilapia lake virus (TiLV). We assessed commercially available probiotic supplements used in TiLV-infected tilapia and performed mortality and cohabitation assays. We developed a mechanistic approach to predict dose-response interactions of probiotic effects on mortality and immune gene response. We used a susceptible-infected-mortality disease model to assess key epidemiological parameters such as transmission rate and basic reproduction number (R0 ) based on our viral load dynamic data. We found that the most marked benefits of probiotics are significantly associated with immune system enhancements (~30%) and reductions in disease transmission (~80%) and R0 (~70%) in tilapia populations, resulting in a higher tolerance of farming densities (~400 fold) in aquaculture. These findings provide early insights as to how probiotic use-related factors may influence TiLV transmission and the immune responses in TiLV-infected tilapia. Our study facilitates understanding the mode of action of probiotics in disease containment and predicting better probiotic dosages in diet and supplements to achieve the optimal culturing conditions. Overall, our analysis assures that further study of rationally designed and targeted probiotics, or mechanistic modelling is warranted on the basis of promising early data of this approach.

Concentration and quantification of Tilapia tilapinevirus from water using a simple iron flocculation coupled with probe-based RT-qPCR.

BackgroundTilapia tilapinevirus, also known as tilapia lake virus (TiLV), is a significant virus that is responsible for the die-off of farmed tilapia across the globe. The detection and quantification of the virus using environmental RNA (eRNA) from pond water samples represents a potentially non-invasive and routine strategy for monitoring pathogens and early disease forecasting in aquaculture systems.MethodsHere, we report a simple iron flocculation method for concentrating viruses in water, together with a newly-developed hydrolysis probe quantitative RT-qPCR method for the detection and quantification of TiLV.ResultsThe RT-qPCR method designed to target a conserved region of the TiLV genome segment 9 has a detection limit of 10 viral copies per µL of template. The method had a 100% analytical specificity and sensitivity for TiLV. The optimized iron flocculation method was able to recover 16.11 ± 3.3% of the virus from water samples spiked with viral cultures. Tilapia and water samples were collected for use in the detection and quantification of TiLV disease during outbreaks in an open-caged river farming system and two earthen fish farms. TiLV was detected from both clinically sick and asymptomatic fish. Most importantly, the virus was successfully detected from water samples collected from different locations in the affected farms (i.e., river water samples from affected cages (8.50 × 103 to 2.79 × 105 copies/L) and fish-rearing water samples, sewage, and reservoir (4.29 × 103 to 3.53 × 104 copies/L)). By contrast, TiLV was not detected in fish or water samples collected from two farms that had previously experienced TiLV outbreaks and from one farm that had never experienced a TiLV outbreak. In summary, this study suggests that the eRNA detection system using iron flocculation, coupled with probe based-RT-qPCR, is feasible for use in the concentration and quantification of TiLV from water. This approach may be useful for the non-invasive monitoring of TiLV in tilapia aquaculture systems and may support evidence-based decisions on biosecurity interventions needed.

Persistent detection of Tilapia lake virus in wild tilapia and tinfoil barbs.

Background and Aim:One of the emerging viral diseases in freshwater fish is Tilapia lake virus (TiLV), which infects all stages of fish and results in mass mortalities. Previously, a TiLV case was detected in the wild environment in Malaysia that involved tilapia and tinfoil barb. Hence, this study aimed to determine the presence of TiLV in wild tilapia (Oreochromis niloticus) as well as tinfoil barbs (Barbonymus schwanenfeldii) at the similar lake after the initial outbreak in year 2017.Materials and Methods:Both fish species were sampled from this lake at a month interval for two years and subjected to TiLV detection using reverse transcriptase-polymerase chain reaction and cell culture isolation. Concurrently, bacterial isolation and water quality measurements were performed to deduce their correlation with TiLV occurrence. Other wild fish species and mollusk were also occasionally sampled during the fish inventory activity at this lake. The fish’s weight, length, and associated clinical signs were noted throughout the entire study period.Results:Mortality was not observed throughout the whole study period, and results indicated a moderate to high prevalence of TiLV infection in both tilapia and tinfoil barbs. There was no correlation between TiLV infection with the isolation rate of opportunistic bacteria such as Aeromonas spp., Plesiomonas spp., and Edwardsiella spp. in the study site. At the same time, the Pearson correlation test revealed a moderate negative correlation between the water pH with the presence of TiLV (R=−0.4472; p<0.05) and a moderate positive correlation between the water iron content with the monthly detection of Aeromonas spp. in wild tilapia. This is contrary to tinfoil barbs, where there was a moderate negative correlation between the water iron content with the monthly isolation of Aeromonas spp. (R=−0.5190; p<0.05). Furthermore, isolation of TiLV on cell culture-induced viral invasion was resulted in the cytopathic effects.Conclusions:Our results suggest that the wild fish may harbor TiLV for an extended period following a massive die-off event in 2017 without any obvious clinical signs and mortality. The persistency of viruses in the wild may need continuous and effective control as well as prevention strategies.

Immunological insights into the resistance of Nile tilapia strains to an infection with tilapia lake virus

The emergence of viral diseases affecting fish and causing very high mortality can lead to the disruption of aquaculture production. Recently, this occurred in Nile tilapia aquaculture where a disease caused by a systemic infection with a novel virus named tilapia lake virus (TiLV) caused havoc in cultured populations. With mortality surpassing 90% in young tilapia, the disease caused by TiLV has become a serious challenge for global tilapia aquaculture. In order to partly mitigate the losses, we explored the natural resistance to TiLV-induced disease in three genetic strains of tilapia which were kept at the University of Göttingen, Germany. We used two strains originating from Nilotic regions (Lake Mansala (MAN) and Lake Turkana (ELM)) and one from an unknown location (DRE). We were able to show that the virus is capable of overcoming the natural resistance of tilapia when injected, providing inaccurate mortality results that might complicate finding the resistant strains. Using the cohabitation infection model, we found an ELM strain that did not develop any clinical signs of the infection, which resulted in nearly 100% survival rate. The other two strains (DRE and MAN) showed severe clinical signs and much lower survival rates of 29.3% in the DRE strain and 6.7% in the MAN strain. The disease resistance of tilapia from the ELM strain was correlated with lower viral loads both at the mucosa and internal tissues. Our results suggest that the lower viral load could be caused by a higher magnitude of a mx1-based antiviral response in the initial phase of infection. The lower pro-inflammatory responses also found in the resistant strain might additionally contribute to its protection from developing pathological changes related to the disease. In conclusion, our results suggest the possibility of using TiLV-resistant strains as an ad hoc, cost-effective solution to the TiLV challenge. However, as the fish from the disease-resistant strain still retained significant virus loads in liver and brain and thus could become persistent virus carriers, they should be used within an integrative approach also combining biosecurity, diagnostics and vaccination measures.\\

Phylogenetic Analysis of Tilapia Lake Virus (TiLV) Isolates from the Philippines Based on Partial Genome Segment 3 Sequences [RESEARCH NOTE

This study aimed to expand current knowledge on TiLV genetic diversity by sequence analysis of a portion of genome segment 3 of TiLV detected from the Philippines. This includes a recently deposited sequence in GenBank (Accession No. LC504279) and six new sequences from cases of infection reported from 2017 to 2020. Phylogenetic analysis of 179 bp fragment of segment 3 showed that the seven TiLV isolates from the Philippines can be divided into three phylogenetic groups. When comparing all sequences, unique nucleotide substitutions and amino acids were noted among these groups. Variation in mortality rates in naturally infected samples was also observed; however, poor environmental conditions during the disease outbreak may also contribute to the mortalities. These suggest the presence of at least three phylogenetic groups of TiLV in the Philippines which has significant implications for the future development of a vaccine, diagnostic kits, and genetic selection programs.

Persistence of Tilapia tilapinevirus in fish rearing and environmental water and its ability to infect cell line.

Tilapia tilapinevirus, or Tilapia Lake Virus (TiLV), is a RNA virus associated with mass morbidity and mortality in tilapia, leading to severe economic losses for global tilapia aquaculture. In this study, we investigated the persistence of TiLV in water by spiking sterile distilled water (SDW), freshwater collected from rearing fish tanks (FW) and natural pond water (PW) at 27°C as a representative of environmental water conditions with 0.6ml of stock virus (3.18×107 viral copies/ml of water). The water samples were filtered through an electronegative charge membrane and quantified using reverse transcriptase quantitative PCR at 0, 3, 5, 7, 10 and 14days post-inoculation. The results revealed that TiLV RNA in SDW was reduced by 1.34 log10 in 14days. A similar approximately 4 log10 removal of the virus in FW and PW was observed at 3 and 7days, respectively. Moreover, the infectivity of TiLV was further studied; the virus lost its infectivity in E-11 cells after 1day in SDW, FW and PW water samples, even though the virus was spiked 10 more times than in the viral persistence study. Taken together, the results could be applied to improving biosecurity practices in tilapia farms by disinfecting or resting reservoir water for at least three to five days prior to stocking tilapia, to limit the spread of TiLV.

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 and show that the disease-free equilibrium is locally asymptotically stable when, . 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, .

Inferring Protein Function in an Emerging Virus: Detection of the Nucleoprotein in Tilapia Lake Virus.

Emerging viruses impose global threats to animal and human populations and may bear novel genes with limited homology to known sequences, necessitating the development of novel approaches to infer and test protein functions. This challenge is dramatically evident in tilapia lake virus (TiLV), an emerging "orthomyxo-like" virus that threatens the global tilapia aquaculture and food security of millions of people. The majority of TiLV proteins have no homology to known sequences, impeding functionality assessments. Using a novel bioinformatics approach, we predicted that TiLV's Protein 4 encodes the nucleoprotein, a factor essential for viral RNA replication. Multiple methodologies revealed the expected properties of orthomyxoviral nucleoproteins. A modified yeast three-hybrid assay detected Protein 4-RNA interactions, which were independent of the RNA sequence, and identified specific positively charged residues involved. Protein 4-RNA interactions were uncovered by R-DeeP and XRNAX methodologies. Immunoelectron microscopy found that multiple Protein 4 copies localized along enriched ribonucleoproteins. TiLV RNA from cells and virions coimmunoprecipitated with Protein 4. Immunofluorescence microscopy detected Protein 4 in the cytoplasm and nuclei, and nuclear Protein 4 increased upon CRM1 inhibition, suggesting CRM1-dependent nuclear export of TiLV RNA. Together, these data reveal TiLV's nucleoprotein and highlight the ability to infer protein functionality, including novel RNA-binding proteins, in emerging pathogens. These are important in light of the expected discovery of many unknown viruses and the zoonotic potential of such pathogens. IMPORTANCE Tilapia is an important source of dietary protein, especially in developing countries. Massive losses of tilapia were identified worldwide, risking the food security of millions of people. Tilapia lake virus (TiLV) is an emerging pathogen responsible for these disease outbreaks. TiLV's genome encodes 10 major proteins, 9 of which show no homology to other known viral or cellular proteins, hindering functionality assessment of these proteins. Here, we describe a novel bioinformatics approach to infer the functionality of TiLV proteins, which predicted Protein 4 as the nucleoprotein, a factor essential for viral RNA replication. We provided experimental support for this prediction by applying multiple molecular, biochemical, and imaging approaches. Overall, we illustrate a strategy for functional analyses in viral discovery. The strategy is important in light of the expected discovery of many unknown viruses and the zoonotic potential of such pathogens.

Immunization of Nile Tilapia (Oreochromis niloticus) Broodstock with Tilapia Lake Virus (TiLV) Inactivated Vaccines Elicits Protective Antibody and Passive Maternal Antibody Transfer.

Tilapia lake virus (TiLV), a major pathogen of farmed tilapia, is known to be vertically transmitted. Here, we hypothesize that Nile tilapia (Oreochromis niloticus) broodstock immunized with a TiLV inactivated vaccine can mount a protective antibody response and passively transfer maternal antibodies to their fertilized eggs and larvae. To test this hypothesis, three groups of tilapia broodstock, each containing four males and eight females, were immunized with either a heat-killed TiLV vaccine (HKV), a formalin-killed TiLV vaccine (FKV) (both administered at 3.6 × 106 TCID50 per fish), or with L15 medium. Booster vaccination with the same vaccines was given 3 weeks later, and mating took place 1 week thereafter. Broodstock blood sera, fertilized eggs and larvae were collected from 6–14 weeks post-primary vaccination for measurement of TiLV-specific antibody (anti-TiLV IgM) levels. In parallel, passive immunization using sera from the immunized female broodstock was administered to naïve tilapia juveniles to assess if antibodies induced in immunized broodstock were protective. The results showed that anti-TiLV IgM was produced in the majority of both male and female broodstock vaccinated with either the HKV or FKV and that these antibodies could be detected in the fertilized eggs and larvae from vaccinated broodstock. Higher levels of maternal antibody were observed in fertilized eggs from broodstock vaccinated with HKV than those vaccinated with FKV. Low levels of TiLV-IgM were detected in some of the 1–3 day old larvae but were undetectable in 7–14 day old larvae from the vaccinated broodstock, indicating a short persistence of TiLV-IgM in larvae. Moreover, passive immunization proved that antibodies elicited by TiLV vaccination were able to confer 85% to 90% protection against TiLV challenge in naïve juvenile tilapia. In conclusion, immunization of tilapia broodstock with TiLV vaccines could be a potential strategy for the prevention of TiLV in tilapia fertilized eggs and larvae, with HKV appearing to be more promising than FKV for maternal vaccination.

Tilapia lake virus disease

This datasheet on Tilapia lake virus disease covers Identity, Distribution.

Dual infections of tilapia parvovirus (TiPV) and tilapia lake virus (TiLV) in multiple tilapia farms: Their impacts, genetic diversity, viral tropism, and pathological effects

Tilapia culture is an essential protein-rich food production sector worldwide. The emergence of pathogens is sometimes associated with fatal outbreaks in tilapia, which dramatically slow production and result in severe economic losses. While tilapia lake virus (TiLV) infection has been associated with fatal diseases in tilapia, the novel tilapia parvovirus (TiPV) has been recently identified and reported to be associated with mass mortality events in tilapia farming. In this study, we identified the coinfection of a novel TiPV and TiLV in multiple independent tilapia farms in Thailand, thus causing significant losses. The full-length genomes of the TiPV were characterized, and a phylogenetic analysis was performed, indicating the genetic diversity of the TiPV and multiple amino acid mutations found in the structural protein VP1. Using the in situ hybridization (ISH) technique, TiPV was localized in the gills, heart, brain, liver, pancreas, spleen, intestine, kidney, eyes, and muscles of tilapia, with evidence of cellular tropism. The TiLV localization was confirmed using a dual ISH/immunohistochemistry protocol and transmission electron microscopy, which indicated a potential association with pathological alterations. Both TiPV and TiLV were successfully propagated in tilapia brain cells and the fish cell line E-11. This study also provided an indication of the potential cellular roles of the pathological features of TiPV coinfection with TiLV in red hybrid tilapia. Since mutations in the parvovirus structural protein led to virulence and extensive infection in susceptible hosts, several point mutations in the VP1 gene with amino acid signatures of TiPV found in tilapia in Thailand warrant future observations. Although the synergism between TiPV and TiLV coinfection remains undetermined, the contributory role of these two viruses requires intensive focus. Further investigations should lead to a suitable strategy for disease control.

Development of a TaqMan quantitative reverse transcription PCR assay to detect tilapia lake virus.

Tilapia lake virus disease (TiLVD) is an emerging viral disease associated with high morbidity and mortality in cultured tilapia worldwide. In this study, we have developed and validated a TaqMan quantitative reverse transcription PCR (RT-qPCR) assay for TiLV, targeting a conserved region within segment 10 of the genome. The RT-qPCR assay was efficient (mean ± SD: 96.71 ± 3.20%), sensitive with a limit of detection of 10 RNA viral copies per reaction, and detected TiLV strains from different geographic regions including North America, South America, Africa, and Asia. The intra- and inter-assay variability ranged over 0.18-1.41% and 0.21-2.21%, respectively. The TaqMan RT-qPCR assay did not cross-react with other RNA viruses of fish, including an orthomyxovirus, a betanodavirus, a picornavirus, and a rhabdovirus. Analysis of 91 proven-positive and 185 proven-negative samples yielded a diagnostic sensitivity of 96.7% and a diagnostic specificity of 100%. The TaqMan RT-qPCR assay also detected TiLV RNA in infected Nile tilapia liver tissue extracts following an experimental challenge study, and it successfully detected TiLV RNA in SSN-1 (E-11 clone) cell cultures displaying cytopathic effects following their inoculation with TiLV-infected tissue homogenates. Thus, the validated TaqMan RT-qPCR assay should be useful for both research and diagnostic purposes. Additionally, the TiLV qPCR assay returns the clinically relevant viral load of a sample which can assist health professionals in determining the role of TiLV during disease investigations. This RT-qPCR assay could be integrated into surveillance programs aimed at mitigating the effects of TiLVD on global tilapia production.

Inferring protein function in an emerging virus: detection of the nucleoprotein in Tilapia Lake Virus

Inferring protein function in an emerging virus: detection of the nucleoprotein in Tilapia Lake Virus