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Abstract: Streptococcosis is an important bacterial disease in Nile tilapia causing severe economic losses to tilapia aquaculture worldwide. The effects of water quality (low‐ [LS] and high‐level [HS] soiling, to mimic clean or dirty surface conditions and temperatures) and disinfectant application (diluted concentrations and exposure time) were characterized on the inactivation of Streptococcus agalactiae isolated from diseased tilapia. Five isolates were tested against three commercial disinfectant products with the main ingredients being povidone iodine (Anidine 100™; AD), benzalkonium chloride (Better BKC 80%™; BKC 80), and a mixture of quaternary ammonium compounds and glutaraldehyde (Chloraldehyde™; CR). CR demonstrated highest efficacy to S. agalactiae inactivation, followed by BKC 80 and AD, respectively. Higher‐level soiling, low temperature, diluted concentrations and short exposure time all decreased the disinfectant efficacy. CR and BKC 80 provided more than 5‐log inactivation at 1‐min exposure at 20°C under HS conditions, and also with ten‐fold‐diluted concentrations at 60‐min exposure time at 30°C. However, AD required 10‐min exposure to effectively remove bacteria under LS conditions at 30°C. The results could facilitate aquaculture management planning that leads to operating cost reductions and improvements in biosecurity. [ABSTRACT FROM AUTHOR]

Abstract: Tilapia lake virus (TiLV) is an emerging pathogen associated with high mortalities of wild and farm‐raised tilapia in different countries. In this study, a SYBR green‐based reverse transcription quantitative polymerase chain reaction (RT‐qPCR) assay targeting segment three of the virus was developed to detect and quantify TiLV in clinical samples and experimentally challenged fish. All 30 field samples with clinical signs and history consistent with TiLV infection were positive for TiLV as detected by the developed RT‐qPCR method. The RT‐qPCR technique provided 100 and 10,000 times more sensitive for virus detection than those offered by the RT‐PCR and virus isolation in cell culture methods, respectively. The detection limit of the RT‐qPCR method was as low as two viral copies/μl. Moreover, the RT‐qPCR technique could be applied for TiLV detection in various fish tissues including gills, liver, brain, heart, anterior kidney and spleen. Significantly, this study delivered an accurate and reliable method for rapid detection of TiLV viruses that facilitates active surveillance programme and disease containment. [ABSTRACT FROM AUTHOR]

Sex identification and the selection of monosex male tilapia are crucial for tilapia aquaculture. In this study, we evaluated the application of ultrasonography as an alternative procedure for sex identification and reproductive assessment in Nile tilapia ( Oreochromis niloticus ). Ultrasonography was performed on 23 fish weighing 232-1,281 g to capture longitudinal and transverse images of the ovaries and testes. Female tilapia were identified by the presence of numerous small egg granules and gray or light gray ovarian tissue and male tilapia by the homogeneous echogenicity and uniform gray tubular appearance of the testes. These distinct echogenic patterns allowed for accurate differentiation between the female and male fish. Ultrasonography achieved a 95% accuracy in sex identification, with minimal stress to the fish during the procedure, compared to manual sex sorting, which demonstrated an accuracy of 87%. Furthermore, the method facilitated the assessment of the reproductive status of the fish, including the maturation stages and fecundity potential. The use of ultrasonography offers significant advantages over traditional methods, such as animal welfare enhancements and improved breeding program efficiency. Our findings support the integration of ultrasonography into aquaculture practices and will promote sustainable and humane fish farming while optimizing reproductive management and productivity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Setthawong, Khemthong, Lertwanakarn and Surachetpong.)

Background and Aim: Immunoglobulin (Ig)Y, a specific type of Ig found in chicken eggs, has potential use in the diagnosis of human and animal diseases. This study assessed the feasibility of using spent laying hens to produce IgY. In addition, the effects of antigen injection on egg and antibody production in new and spent laying hens were compared., Materials and Methods: Hens were intramuscularly injected with three booster shots of antigens. IgY was extracted from egg yolks using polyethylene glycol 6000 precipitation followed by dialysis., Results: Spent laying hens (83 weeks) consistently showed lower egg production than new laying hens (27 weeks) throughout the study. Post-immunization, a further decline in egg production was observed in spent laying hens, and egg production stopped after the second antigen injection. Eggs from spent laying hens were less dense than eggs from new hens. Despite lower IgY levels in eggs from spent laying hens, the heavy-to-light chain ratio remained consistent in both groups. Notably, IgY from spent and new laying hens demonstrated effective hemagglutination against cat erythrocytes in the A blood group., Conclusions: This study demonstrated the potential of using spent laying hens to produce IgY, with significant implications for future research, immunotherapy, and diagnostic applications, despite the observed reduction in egg production compared with new laying hens., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Metheenukul, et al.)

Tilapia Lake Virus (TiLV) is associated with pathological changes in the brain of infected fish, but the mechanisms driving the virus's neuropathogenesis remain poorly characterized. TiLV establishes a persistent infection in the brain of infected fish even when the virus is no longer detectable in the peripheral organs, rendering therapeutic interventions and disease management challenging. Moreover, the persistence of the virus in the brain may pose a risk for viral reinfection and spread and contribute to ongoing tissue damage and neuroinflammatory processes. In this review, we explore TiLV-associated neurological disease. We discuss the possible mechanism(s) used by TiLV to enter the central nervous system (CNS) and examine TiLV-induced neuroinflammation and brain immune responses. Lastly, we discuss future research questions and knowledge gaps to be addressed to significantly advance this field.

The guppy, Poecilia reticulata, is one of the most common cultured ornamental fish species, and a popular pet fish highly desired by hobbyists worldwide due to its availability of many brilliantly coloured fish of many varieties. The susceptibility of guppies to diseases presents a remarkable concern for both breeders and hobbyists. In this study, we report the emergence of disease in fancy guppies caused by a previously uncharacterized virus in the USA. This virus was isolated from moribund guppies in two separate outbreaks in California and Alabama, from December 2021 to June 2023. The infected guppies presented with acute morbidity and mortality shortly after shipping, displaying nonspecific clinical signs and gross changes including lethargy, anorexia, swimming at the water surface, gill pallor, mild to moderate coelomic distension and occasional skin lesions including protruding scales, skin ulcers and hyperaemia. Histological changes in affected fish were mild and nonspecific; however, liver and testes from moribund fish were positive for Tilapia lake virus (TiLV), the single described member in the family Amnoonviridae, using immunohistochemistry and in situ hybridization, although the latter was weak. A virus was successfully recovered following tissue inoculation on epithelioma papulosum cyprini and snakehead fish cell lines. Whole genome sequencing and phylogenetic analyses revealed nucleotide and amino acid homologies from 78.3%-91.2%, and 78.2%-97.7%, respectively, when comparing the guppy virus genomes to TiLV isolates. Based on the criteria outlined herein, we propose the classification of this new virus, fancy tailed guppy virus (FTGV), as a member of the family Amnoonviridae, with the name Tilapinevirus poikilos (from the Greek 'poikilos', meaning of many colours; various sorts, akin to 'poecilia')., (© 2024 The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd.)

Tilapia lake virus (TiLV) presents a substantial threat to global tilapia production. Despite the development of numerous cell lines for TiLV isolation and propagation, none have been specifically derived from red hybrid tilapia ( Oreochromis spp.). In this study, we successfully established a new cell line, RHTiB, from the red hybrid tilapia brain. RHTiB cells were cultured for 1.5 years through over 50 passages and demonstrated optimal growth at 25 °C in Leibovitz-15 medium supplemented with 10% fetal bovine serum at pH 7.4. Morphologically, RHTiB cells displayed a fibroblast-like appearance, and cytochrome oxidase I gene sequencing confirmed their origin from Oreochromis spp. Mycoplasma contamination testing yielded negative results. The revival rate of the cells post-cryopreservation was observed to be between 75 and 80% after 30 days. Chromosomal analysis at the 25th passage revealed a diploid count of 22 pairs (2n = 44). While no visible cytopathic effects were observed, both immunofluorescence microscopy and RT-qPCR analysis demonstrated successful TiLV propagation in the RHTiB cell line, with a maximum TiLV concentration of 10 7.82 ± 0.22 viral copies/400 ng cDNA after 9 days of incubation. The establishment of this species-specific cell line represents a valuable advancement in the diagnostic and isolation tools for viral diseases potentially impacting red hybrid tilapia.

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., Competing Interests: The authors declare there are no competing interests., (©2023 Raksaseri et al.)

Amnoonviridae is a family of negative-sense RNA viruses with genomes totalling about 10.3 kb. These viruses have been found in fish. The amnoonvirid genome consists of 10 segments, each with at least 1 open reading frame (ORF). The RNA1-3 ORFs encode the three subunits of the viral polymerase. The RNA4 ORF encodes a nucleoprotein. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Amnoonviridae , which is available at ictv.global/report/amnoonviridae.

Background and Aim: The association between bacterial DNA in stifle joints, including those with cranial cruciate ligament rupture (CCLR) and medial patellar luxation (MPL), and osteoarthritis in dogs remains elusive. This study investigated the potential association between the detection of bacterial DNA and osteoarthritis in dogs using a broad-range polymerase chain reaction technique targeting the 16S ribosomal RNA gene., Materials and Methods: Synovial fluid (35 samples) and knee tissue samples (32 samples) were obtained from 35 dogs diagnosed with CCLR (n = 20; 11 males and nine females) or MPL (n = 15; five males and 10 females) who underwent a surgical operation between October 2014 and April 2015., Results: Dogs with CCLR had a higher average osteoarthritis score than those with MPL (2.0 ± 0.9 vs. 0.5 ± 0.9; p = 0.005). Bacterial DNA was detected in the stifle joints of 60.71% of dogs with MPL. Pelomonas spp. (25.00%), Halomonas spp. (17.86%), and 5 other species (17.86%) were the most frequently identified bacteria. Bacterial DNA was detected in 41.03% of dogs with CCLR. Pelomonas spp. (15.38%), Sphingomonas spp. (10.26%), Halomonas spp. (5.13%), and 4 other species (10.26%) were the most frequently identified bacteria. No significant difference was observed in the prevalence of bacterial DNA obtained from tissue samples (46.88%) or joint fluid samples (51.43%). The presence of bacterial DNA was not associated with the type of knee injury (MPL or CCLR; p = 1.000). There was a higher prevalence of bacterial DNA in samples from dogs with moderate-to-severe osteoarthritis (94.44%) than in those with minimal osteoarthritis (41.18%), and a significant association between the presence of bacterial DNA and moderate-to-severe osteoarthritis was identified (p < 0.01)., Conclusion: Dogs with moderate-to-severe osteoarthritis were more likely to have bacterial DNA in their stifle joints than those with no or minimal osteoarthritis. These findings provide valuable insight into the potential role of bacterial DNA in joint tissue or joint fluid and the development of osteoarthritis in dogs., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Tuek-Um, et al.)

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., Competing Interests: Declaration of competing interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Tilapia lake virus (TiLV) causes high mortality in farmed and wild tilapia in various countries. We developed a highly specific and sensitive droplet digital polymerase chain reaction (ddPCR) assay to detect and quantify TiLV. The ddPCR assay could detect the virus at a lower threshold than the reverse transcription-quantitative polymerase reaction (RT-qPCR) method, and the sensitivity of the ddPCR assay was 10-fold higher. The diagnostic sensitivity and specificity of the ddPCR assay were 100% and did not cross-react with tilapia tissues infected with Tilapia parvovirus, Infectious spleen and kidney necrosis virus, Aeromonas hydrophila, Streptococcus agalactiae, S. iniae and Francisella noatunensis. The assay reproducibility was demonstrated by a high correlation coefficient of 0.998, and the inter-assay coefficients of variability indicated that the ddPCR assay exhibited low variability within and between measurements. The detection limit of the TiLV ddPCR assay was 100 fg cDNA, which is equal to 3.3 copies of TiLV. Furthermore, the ddPCR assay could detect TiLV in mucus, water and infected tissue samples and the lowest copy number of TiLV detected in water samples by the ddPCR assay was 7.9 ± 0.99 copies/reaction The results of the clinical samples tested for TiLV revealed that the ddPCR assay had a relatively higher detection rate than the RT-qPCR method. Overall, the ddPCR method offers a highly promising approach for the absolute quantification of TiLV in carrier fish and samples from the environment with low viral concentrations., (© 2023 John Wiley & Sons Ltd.)

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., Competing Interests: The authors declare that there are no financial and non-financial conflicts of interest. The indicators and methodology for assessing fish welfare is already in public domain, as per previous publication, with Ana Silvia being the main author, as part of her Ph.D. Murilo Quintiliano and Pau Badia are FAI employees and contributed for the paper. Ana Silvia is a WAI employee and has also contributed for the paper. The data belong to the corresponding author. The purpose of the project was to evaluate the status of tilapia welfare in Thailand without any financial support from any private company.

The giant snakehead, Channa micropeltes, is an increasingly important economic freshwater fish in Thailand and other regions of Asia. Presently, giant snakehead are cultured under intensive aquaculture conditions, leading to high stress and conditions favouring disease. In this study, we reported a disease outbreak in farmed giant snakehead with a cumulative mortality of 52.5%, continuing for 2 months. The affected fish exhibited signs of lethargy, anorexia and haemorrhage of the skin and eyes. Further bacterial isolations revealed two different types of colonies on tryptic soy agar: small white, punctate colonies of gram-positive cocci and cream-coloured, round and convex colonies of rod-shaped gram-negative bacteria. Additional biochemical and species-specific PCR analysis based on 16S rRNA confirmed the isolates as Streptococcus iniae and Aeromonas veronii. Multilocus sequence analysis (MLSA) placed the S. iniae isolate into a large clade of strains from clinically infected fish worldwide. Gross necropsy findings showed liver congestion, pericarditis and white nodules in the kidney and liver. Histologically, the affected fish showed focal to multifocal granulomas with inflammatory cell infiltration in kidney and liver, enlarged blood vessels with mild congestion within the meninges of the brain and severe necrotizing and suppurative pericarditis with myocardial infarction. Antibiotic susceptibility tests revealed that S. iniae was sensitive to amoxicillin, erythromycin, enrofloxacin, oxytetracycline, doxycycline and resistant to sulfamethoxazole-trimethoprim, while the A. veronii was susceptible to erythromycin, enrofloxacin, oxytetracycline, doxycycline, sulfamethoxazole-trimethoprim and resistant to amoxicillin. Conclusively, our findings highlighted the natural concurrent bacterial infections in cultured giant snakehead, which support the implementation of appropriate treatment and control strategies., (© 2023 John Wiley & Sons Ltd.)

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., (© 2023 John Wiley & Sons Ltd.)

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), an enveloped negative-sense single-stranded RNA virus, causes tilapia lake virus disease (TiLVD), which is associated with mass mortality and severe economic impacts in wild and farmed tilapia industries worldwide. In this study, we developed a chitosan nanoparticle TiLV immersion vaccine and assessed the efficacy of the vaccine in laboratory and field trials. Transmission electron microscopy showed that the inactivated vaccine had a particle size of 210.3 nm, while the nano inactivated vaccine had a spherical shape with a diameter of 120.4 nm. Further analysis using fluorescent staining and immunohistochemistry analysis revealed the mucoadhesive properties of the nanovaccine (CN-KV) through fish gills. We assessed the efficacy of an immersion-based TiLV nanovaccine using a cohabitation challenge model. The fish that received the nanovaccine showed better relative percent survival (RPS) at 68.17% compared with the RPS of the inactivated virus vaccine (KV) group at 25.01%. The CN-KV group also showed a higher TiLV-specific antibody response than the control and KV groups (p < 0.05). Importantly, under field conditions, the fish receiving the CN-KV nanovaccine had better RPS at 52.2% than the nonvaccinated control group. Taken together, the CN-KV nanovaccinated fish showed better survival and antibody response than the control and KV groups both under laboratory control challenge conditions and field trials. The newly developed immersion-based nanovaccine is easy to administer in small fish, is less labor-intensive, and allows for mass vaccination to protect fish from TiLV infection., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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 (R 0 ) 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 R 0 (~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., (© 2022 John Wiley & Sons Ltd.)

Feline morbillivirus-1 (FeMV-1) is a viral pathogen associated with kidney disease in domestic cats and wild felids. We initially identified the FeMV-1 from the lung of a necropsied dog with severe pulmonary disease by the reverse transcription polymerase chain reaction (RT-PCR). Thereafter, we investigated FeMV-1 in nasal and oral swab samples from 73 healthy and 113 dogs with respiratory illnesses. We found polymerase chain reaction (PCR)-positive FeMV-1 from only 14/113 (12.39%) dogs with respiratory disease (p = .001). Of these 14 dogs, six were co-infected with other canine respiratory viruses (6/14; 42.86%). Two independent immunohistochemistry procedures, using antibodies against matrix and phosphoprotein of FeMV-1, confirmed the presence of FeMV-1 in lung tissues of two necropsied dogs (out of a total of 22 dogs, 9.09%) that died from respiratory disease. This finding corresponded to transmission electron microscopy findings that paramyxoviral particles exist in lung epithelia. FeMV-1 antigen localization was also evident in the kidney, lymphoid and brain tissues of two deceased dogs. FeMV-1 was successfully isolated from a necropsied dog and from two living dogs, all with respiratory illnesses, which supports FeMV infection in dogs. The detection of FeMV-1 in dog tissues expands the known tropism of this virus to a non-felid host. Our findings indicate that FeMV-1, alone or in co-infection with other viral pathogens, might contribute to respiratory illness and death in dogs., (© 2021 Wiley-VCH GmbH.)

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 0.6 ml of stock virus (3.18 × 10 7 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 14 days post-inoculation. The results revealed that TiLV RNA in SDW was reduced by 1.34 log 10 in 14 days. A similar approximately 4 log 10 removal of the virus in FW and PW was observed at 3 and 7 days, respectively. Moreover, the infectivity of TiLV was further studied; the virus lost its infectivity in E-11 cells after 1 day 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., (© 2022 John Wiley & Sons Ltd.)

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.\., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Tilapia parvovirus (TiPV) is a novel parvovirus associated with high mortality in Nile tilapia and red hybrid tilapia, leading to severe economic losses for tilapia aquaculture. It is critical to develop a sensitive and accurate assay to detect TiPV in fish tissues. In this study, new TaqMan probe-based quantitative PCR (qPCR) assays targeting the non-structural (NS) and viral protein (VP) genes of TiPV were developed. The standard curves of the assays were 95.64%-98.96% over a wide linear range of 10 9 -10 1 copies of the corresponding standard DNA per reaction. The intra- and inter-assay coefficients of variation were in the ranges 0.54%-2.50% and 0.13%-1.17%, respectively, which suggests good repeatability and reproducibility. The detection limit of the TaqMan TiPV assays was 10 copies/µl. The application of the TaqMan qPCR assays to field samples revealed that they had comparable sensitivity to a previously developed SYBR Green qPCR, but more sensitive than the conventional PCR. No cross-reactivity of the TaqMan TiPV assays was found with the samples infected with other viruses and bacteria. Overall, the assays offered high sensitivity and specificity in the detection of low concentrations of TiPV DNA in infected tilapia samples. These new TaqMan qPCR assays could provide a valuable diagnostic tool for the reliable and specific detection of TiPV in experimental and field samples., (© 2021 John Wiley & Sons Ltd.)

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.

The recently discovered Tilapia parvovirus (TiPV) was the first Parvovirus confirmed to infect fish, causing mortality outbreaks in farmed adult Nile tilapia in China. Severe mortality outbreaks caused by Tilapia tilapinevirus (TiLV) to farmed tilapia in Thailand revealed the concomitant occurrence of TiPV. Out of ten fish farms screened, TiPV was detected in one site rearing juvenile red hybrid tilapia. Clinical signs included abnormal swimming, scale protrusion, skin and muscle haemorrhaging, exophthalmia and generalized anaemia. Histological findings showed extensive infiltration of lymphocytes, with increased melanomacrophage centres in the anterior kidney and spleen, erythrocyte depletion in the spleen and hepatic syncytial cells. Both TiLV and TiPV were systemically distributed in the body of moribund fish. The analysis of the near-complete TiPV genome isolated from Thailand revealed 98.74% sequence identity to the formerly isolated from China, together with a highly conserved and comparable genomic organization and with a 3 nucleotides deletion in the 5-UTR. The viral genome structure was highly conserved for each of its components, with nucleotide and amino acid identity ranging from 100% for ORF1 to 97% for ORF2, and with conserved HuH and Walker loop motifs within NS1. Taken together, our results document the first detection of TiPV outside China, thus for the first time in Thailand. Moreover, TiPV was detected for the first time during a natural occurrence in farmed red hybrid tilapia and involved in co-infection pattern with TiLV. Diagnostic investigations during tilapia disease outbreaks should include the screening for TiPV. Further studies are needed to elucidate TiPV genomic variance, pathobiology, including focussing on the outcomes of TiLV-TiPV co-infection patterns, necessary to enable risk assessment for the worldwide spreading of TiPV and to design adequate control measures against these emerging viruses in tilapia., (© 2021 Wiley-VCH GmbH.)

Tilapia lake virus (TiLV) is a highly contagious novel orthomyxo-like RNA virus that is negatively impacting tilapia production worldwide. To prevent TiLV from spreading globally, the infection status of source farms needs to be established prior to the movement of live tilapia to minimize the risk of horizontal transmission. However, testing individual fish for TiLV requires large sample sizes, when within-farm prevalence is low and is costly, time-consuming, and labour-intensive. The objective of the present study was to evaluate the use of pool testing for TiLV detection and to estimate within-farm prevalence based on the percentage of positive pooled samples. Pooled samples of liver and spleen were prepared by diluting different numbers of positive tissue samples with negative homogenate tissue samples. A tissue pool from 5 or 10 individual fish containing at least one TiLV-positive sample was sufficient to yield a positive result except when cycle threshold (Ct) values were between 31 and the cut-off value of 34. Additionally, our study characterized viral load in two farms after TiLV outbreaks. Bayesian modelling showed that within-farm prevalence could be estimated from the percentage of positive pools of size 5 using prior information about pool sensitivity and specificity, and prevalence, and assuming random sampling of tilapia from infected ponds. Ninety-five percent posterior intervals for prevalence were slightly wider than those obtained based on the results of individual samples. Findings in the present study corroborate the use of a pooling strategy for post-outbreak surveillance of TiLV., (© 2020 Wiley-VCH GmbH.)

In mammals, the relationship between the immune system and behavior is widely studied. In fish, however, the knowledge concerning the brain immune response and behavioral changes during brain viral infection is very limited. To further investigate this subject, we used the model of tilapia lake virus (TiLV) infection of zebrafish ( Danio rerio ), which was previously developed in our laboratory. We demonstrated that TiLV persists in the brain of adult zebrafish for at least 90 days, even when the virus is not detectable in other peripheral organs. The virions were found in the whole brain. During TiLV infection, zebrafish displayed a clear sickness behavior: decreased locomotor activity, reduced food intake, and primarily localizes near the bottom zone of aquaria. Moreover, during swimming, individual fish exhibited also unusual spiral movement patterns. Gene expression study revealed that TiLV induces in the brain of adult fish strong antiviral and inflammatory response and upregulates expression of genes encoding microglia/macrophage markers. Finally, using zebrafish larvae, we showed that TiLV infection induces histopathological abnormalities in the brain and causes activation of the microglia which is manifested by changes in cell shape from a resting ramified state in mock-infected to a highly ameboid active state in TiLV-infected larvae. This is the first study presenting a comprehensive analysis of the brain immune response associated with microglia activation and subsequent sickness behavior during systemic viral infection in zebrafish., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mojzesz, Widziolek, Adamek, Orzechowska, Podlasz, Prajsnar, Pooranachandran, Pecio, Michalik, Surachetpong, Chadzinska and Rakus.)

Tilapia lake virus (TiLV) is a notable contagious agent that causes massive economic losses in the tilapia industry globally. Evaluations of the histological changes associated with TiLV infection are not only crucial for diagnosis, but also to gain an understanding of the disease. We therefore synthesized a rabbit polyclonal immunoglobulin G antibody against TiLV and developed an immunohistochemical (IHC) procedure to detect TiLV localization in the tissues of infected fish for comparison with in situ hybridization (ISH) testing. A total of four different sample cohorts derived from TiLV-infected fish was used to validate the IHC procedure. The TiLV IHC application was successfully developed and facilitated nuclear and cytoplasmic immunolabelling in the intestines, gills, brain, liver, pancreas, spleen, and kidneys that corresponded with the ISH results. Apart from the ISH results, TiLV-IHC signals were clearly evident in the endothelial cells of various organs, the circulating leukocytes in the blood vessels, and the areas of tissue inflammation. Among the tested sample cohorts, the intestines, gills, and brain had IHC-positive signals, highlighting the possibility of these organs as common TiLV targets. Immunological staining pattern and distribution corresponded with the TiLV viral load but not the inoculation route. The TiLV IHC was also capable of detecting TiLV infection in the experimentally challenged ornamental cichlids, Mozambique tilapia, giant gourami, and naturally infected tilapia, indicating the dynamic range of IHC for TiLV detection. Overall, our study delivers the first IHC platform to detect TiLV infection and provides novel evidence of cellular tropism during TiLV infection. Our findings also reveal the TiLV distribution pattern of infected fish and propose the endotheliotropism and lymphotropism of this virus, which requires further elaboration. Importantly, this new IHC procedure could be applied to study the pathogenesis and interaction of TiLV in future research., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The emergence of tilapia lake virus (TiLV) has had a severely negative impact on global tilapia aquaculture. TiLV infection has been reported at different life stages of tilapia, with more emphasis on fry and fingerlings; however, the virulence and pathology of TiLV at different tilapia size remains unexplored. In this study, tilapias from a single spawning were raised to 5 g, 25 g, and 65 g, and subsequently challenged by the intraperitoneal injection and cohabitation of a virulent strain of TiLV. The cumulative mortality, viral load, and histopathology of the fish were determined until 22 days post-infection (dpi). The cumulative mortality of the 5 g, 25 g, and 65 g fish was 85% (±1.67), 55% (±2.89), and 51.67% (±7.49), respectively. At 14 dpi, the mean TiLV load in the liver of the 5 g fish was significantly higher than in the 25 g and 65 g fish. All the weight groups showed severe pathological changes in the liver, spleen, and intestine after TiLV infection, but no particular difference was otherwise noted during the study with the exception of higher pathological scores in the liver of the small fish at 14 dpi. Overall, this study indicated that small fish are more susceptible to TiLV infection than large fish. Although multiple factors, including environmental factors, farm management practices, strains of virus could contribute to different susceptibility of fish to viral infection, the present study provides the evidence to support that fish weight affects the mortality and clinical outcome during TiLV infection. More intensive measures such as strict biosecurity and disease surveillance during the susceptible weight should therefore be emphasized to reduce the impact of this virus., Competing Interests: The authors declare that they have no competing interests., (© 2021 Roy et al.)

Tilapia tilapinevirus, or tilapia lake virus (TiLV), is a highly contagious virus found in tilapia and its hybrid species that has been reported worldwide, including in Asia, the Americas, and Africa. In this study, we experimentally challenged Mozambique tilapia ( Oreochromis mossambicus ) with a virulent TiLV strain, VETKU-TV01, at both low (1 × 10 3 TCID 50 /mL) and high (1 × 10 5 TCID 50 /mL) concentration. After the challenge, the Mozambique tilapia showed pale skin with some hemorrhage and erosion, lethargy, abdominal swelling, congestion around the eye, and exophthalmos; there was a cumulative mortality rate at 48.89% and 77.78% in the groups that received the low and high concentration, respectively. Quantitative PCR and in situ hybridization confirmed the presence of TiLV in the internal organs of moribund fish. Notably, severe histopathological changes, including glycogen depletion, syncytial hepatic cells containing multiple nuclei and intracytoplasmic inclusion bodies, and infiltration of melanomacrophage into the spleen, were frequently found in the Mozambique tilapia challenged with high TiLV concentration. Comparatively, the infectivity and pathology of the TiLV infection in Mozambique tilapia and red hybrid tilapia ( Oreochromis spp.) were found to be similar. Our results confirmed the susceptibility of Mozambique tilapia, which has recently been determined to be a vulnerable species, to TiLV infection, expanding knowledge that the virus can cause disease in this fish species.

Tilapia lake virus (TiLV; genus: Tilapinevirus, family: Amnoonviridae) is a recently characterised enveloped virus with a linear, negative-sense single-stranded RNA genome, which causes high mortality in tilapia species. In the present study, we demonstrated that zebrafish (Danio rerio) larvae are susceptible to TiLV infection upon systemic injection. TiLV replicated in zebrafish larvae and caused their high mortality (of about 70%). Histopathological examination revealed that TiLV infection caused pathological abnormalities in zebrafish larvae that were well visible within the brain. Moreover, gene expression analysis revealed that TiLV infection induced up-regulation of the expression of the immune-related genes encoding pathogen recognition receptors involved in sensing of viral dsRNA (rig-I (ddx58), tlr3, tlr22), transcription factors (irf3, irf7), type I interferon (infϕ1), antiviral protein (mxa), and pro-inflammatory cytokine (il-1β). We also demonstrated the protective role of the recombinant zebrafish IFNϕ1 on the survival of zebrafish larvae during TiLV infection. Our results show the importance of type I IFN response during TiLV infection in zebrafish larvae and demonstrate that zebrafish is a good model organism to study interactions between TiLV - a newly emerging in aquaculture virus, and fish host., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Tilapia lake virus (TiLV) is regarded as one of the most important pathogens in tilapia aquaculture worldwide. Despite this, little is known regarding disease pathogenesis and immune responses to infection. The main objective of this study was to investigate the tissue distribution, histopathological changes, and immune response of fish exposed to TiLV. Nile tilapia (Oreochromis niloticus) maintained at 25 ± 2 °C were challenged with TiLV via intragastric-gavage. At 0.5, 1, 3, 5, 7, 10 and 15 days post-challenge (dpc), six fish per treatment were euthanized and subjected to complete necropsy. TiLV exposed fish presented 45% cumulative mortality at the end of the study. Gross lesions included cutaneous petechiae and ecchymoses, scale losses, skin ulcers, and exophthalmia. Mild multifocal hepatocellular degeneration and necrosis was observed as early as 3 dpc occasionally accompanied by syncytial formation, intracytoplasmic inclusion bodies, and inflammatory infiltrates of lymphocytes at subsequent time points. Necrosis of epithelial cells of the gastric glands and intestinal glands was also observed as early as 5 dpc. Intestinal samples showed reactive in situ hybridization signals as early as 1 dpc. No other lesions were observed in the brain or other organs. Histological changes were associated with viral dissemination and disease progression, as evidenced by increased TiLV detection in the intestine, gills, liver and spleen. Highest TiLV abundance was detected 7 dpc in gills, intestine, and liver showing an average of 6 LOG genome equivalent per ng of total RNA. Different transcript abundance was detected for the pro-inflammatory cytokine interleukin-1β and interferon-induced myxovirus resistance protein gene in the mucosal sites (gills and intestine). Interferon regulatory transcription factor 3 transcript was more abundant in systemic organs (liver and spleen) while the expression in gills and intestine showed mixed expression at different time points. On the other hand, transforming growth factor β expression patterns differed amongst the tissues with a trend towards downregulation of the gene in liver and gills, and a trend towards upregulation in the spleen and intestine. Overall, these results demonstrate the intestinal routes as a main port of entry for TiLV, which subsequently spreads systematically throughout the fish body., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Tilapia lake virus (TiLV) causes an emerging viral disease associated with high mortality and economic damage in tilapia farming around the world. The use of probiotics in aquaculture has been suggested as an alternative to antibiotics and drugs to reduce the negative impact of bacterial and viral infections. In this study, we investigate the effect of probiotic Bacillus spp. supplementation on mortality, viral load, and expression of immune-related genes in red hybrid tilapia ( Oreochromis spp.) upon TiLV infection. Fish were divided into three groups, and fed with: control diet, 0.5% probiotics-supplemented diet, and 1% probiotics-supplemented diet. After 21 days of experimental feeding, the three groups were infected with TiLV and monitored for mortality and growth performances, while organs were sampled at different time points to measure viral load and the transcription modulation of immune response markers. No significant difference was found among the groups in terms of weight gain (WG), average daily gain (ADG), feed efficiency (FE), or feed conversion ratio (FCR). A lower cumulative mortality was retrieved from fish fed 0.5% and 1% probiotics (25% and 24%, respectively), compared to the control group (32%). Moreover, fish fed with 1% probiotic diet had a significantly lower viral load, than those fed with 0.5% probiotic and control diet at 5, 6, 9, and 12 days post infection-challenge (dpc). The expression patterns of immune-related genes, including il-8 (also known as CXCL8), ifn-γ , irf-3 , mx , rsad-2 (also known as VIPERIN) showed significant upregulation upon probiotic treatment during the peak of TiLV pathogenesis (between 9 and 12 dpc) and during most of the study period in fish fed with 1% probiotics-supplemented diet. Taken together, these findings indicate that dietary supplementation using Bacillus spp. probiotics may have beneficial effects to strengthen tilapia immunity and resistance against TiLV infections. Therefore, probiotic treatments may be preventively administered to reduce losses caused by this emerging viral infection in tilapia aquaculture.

Tilapia lake virus (TiLV) is an emerging virus associated with high mortality in cultured tilapia. Since the first report of tilapia lake virus, it has been detected in diseased tilapia in sixteen countries around the world. Thus, there is an urgent need to develop an efficacious vaccine to prevent TiLV disease (TiLVD) and reduce its global economic impact. Understanding the role of the adaptive immune response following exposure of tilapia to TiLV is a critical step in the development of such a vaccine. In this study, we challenged red hybrid tilapia by cohabitation or intraperitoneal injection and demonstrated that surviving fish develop a protective immunity. We also demonstrated that tilapia that survived experimental infections possess significant antibodies against the protein encoded by the TiLV segment 4. We then developed a TiLV indirect ELISA to determine the antibody response in tilapia. The ELISA revealed high antibody levels in survivors of experimental challenges and following outbreaks on farms. The ELISA effectively distinguished TiLV-exposed from unexposed tilapia and was used to monitor anti-TiLV antibody kinetics following infection. During the primary infection, tilapia developed an antibody response as early as 7 days post TiLV challenge (dpc), peaked at 15 dpc, showed a gradual decline up until about 42 dpc, but persisted in some fish up until day 110 dpc. Upon re-infection, an increased antibody response occurred within 7-14 days, demonstrating that tilapia that survive TiLV infections develop humoral memory. In conclusion, our results demonstrated that tilapia mount antibody responses against TiLV that supports protective immunity to subsequent TiLV disease. The persistence of anti-TiLV antibodies in survivors following a single exposure suggests a single vaccination might be adequate to protect tilapia during the entire grow-out period. This study provides important information about the immune response of tilapia following exposure to TiLV as a first step in the development of an efficacious vaccine against this emerging and economically important viral disease., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Tilapia lake virus (TiLV) is a highly contagious pathogen that has detrimental effects on tilapia farming. This virus was discovered in 2014 and has received tremendous global attention from the aquaculture sector due to its association with high fish mortalities and its strong economic impact on the tilapia aquaculture industry. Currently, TiLV has been reported in 16 countries, and this number is continuing to rise due to improved diagnostic assays and surveillance activities around the world. In this review, we summarize the up-to-date knowledge of TiLV with regard to TiLV host species, the clinical signs of a TiLV infection, the affected tissues, pathogenesis and potential disease risk factors. We also describe the reported information concerning the virus itself: its morphology, genetic make-up and transmission pathways. We review the current methods for virus detection and potential control measures. We close the review of the TiLV story so far, by offering a commentary on the major TiLV research gaps, why these are delaying future TiLV research and why the TiLV field needs to come together and proceed as a more collaborative scientific community if there is any hope limiting the impact of this serious virus., (© 2020 John Wiley & Sons Ltd.)

Background and Aim: MicroRNAs (miRNAs) are responsible for gene expression control at the post-transcription level in many species. Several miRNAs are required in the regulation of immune responses, such as B-cell differentiation, T-cell receptor signaling pathway, CD4 + T cell selection, and so on. Studies on miRNAs have been extensively conducted in humans and mice; however, reports relevant to miRNAs, especially miR-155 and miR-181, in pigs are limited. Consequently, the present study aimed to investigate the structures, target genes, and expressions of miR-155 and miR-181 in various porcine cells and tissues., Materials and Methods: Five healthy male pigs from a porcine reproductive and respiratory syndrome virus-negative farm were studied. Before slaughter, blood samples were collected for peripheral blood mononuclear cell isolation. After slaughter, samples of spleen, lymph nodes, and forelimb muscles were collected. Both miR-155 and miR-181 were investigated for their structures with RNAfold web server, for their target genes from three online web servers, and for their expressions using polymerase chain reaction (PCR)., Results: The structures of miR-155 and miR-181 contained hairpins with free energies of -35.27 and -35.29 kcal/mole, respectively. Target gene prediction revealed that miR-155 had perfect complementarity with Socs1 and Mapk3k14 , while miR-181 had perfect complementarity with Ddx3x, Nfat5, Foxp1 , and Mpp5 . PCR showed that both miRNAs were detectable from all investigated cells and tissues. Moreover, the highest expression of both miRNAs was found from the lymph node of the pigs., Conclusion: Both miR-155 and miR-181 might be involved with the regulation of porcine immune functions as both miRNAs were detected in several cells and tissues of the pigs. In addition, they had very high complementarities with the seed regions of several immune-related genes., (Copyright: © Ninsuwon, et al.)

Tilapia lake virus (TiLV) is a novel enveloped orthomyxo-like virus with a genome of 10 segments of linear negative-sense single-stranded RNA. It causes massive mortality of wild and farmed tilapia species and because of its spread in Asia, Africa, South and North America, it is considered a threat to tilapia aquaculture. Here, we have evaluated the possible use of zebrafish (Danio rerio) to study immune response and host-pathogen interactions during an infection with TiLV. Adult zebrafish were infected with TiLV by intraperitoneal (i.p) injection or by cohabitation. Increased viral load was observed in liver, spleen and kidney of i.p. injected fish at 1, 3, 6, and 14 days post infection (dpi) but not in fish from the cohabitation group (only liver was tested). We also demonstrated that in spleen and kidney i.p. injection of TiLV induced up-regulation of the expression of the immune-related genes encoding pathogen recognition receptors involved in sensing of viral dsRNA (rig-I, tlr3, tlr22), transcription factors (irf3, irf7), type I interferon (infϕ1), antiviral protein (mxa), pro-inflammatory (il-1β, tnf-α, il-8, ifnγ1-2) and anti-inflammatory (il-10) cytokines, CD4 markers (cd4-1, cd4-2), and IgM (igm). Moreover, tissue tropism of TiLV and histopathological changes were analyzed in selected organs of i.p. injected zebrafish. Our results indicate that zebrafish is a good model to study mechanisms of the TiLV infection and to follow antiviral responses., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the aquaculture industry that has resulted in the mass morbidity and mortality of tilapia in many parts of the world. An effective, rapid, and accurate diagnostic assay for TiLV infection is therefore necessary to detect the initial infection and to prevent the spread of the disease in aquaculture farming. In this study, a highly sensitive and practical reverse transcription loop-mediated isothermal amplification (RT-LAMP) method is presented to detect tilapia lake virus in fish tissue. A comparison of the RT-qPCR and RT-LAMP assays of infected samples revealed positive results in 63 (100%) and 51 (80.95%) samples, respectively. Moreover, an analysis of uninfected samples showed that all 63 uninfected tissues yielded negative results for both the RT-qPCR and RT-LAMP assays. The cross-reactivity with five pathogens in tilapia was evaluated using RT-LAMP, and all the tests showed negative results. Both the liver and mucus samples obtained from infected fish showed comparable results using the RT-LAMP method, suggesting that mucus can be used in RT-LAMP as a nonlethal assay to avoid killing fish. In conclusion, the results demonstrated that the presented RT-LAMP assay provides an effective method for TiLV detection in tilapia tissue within 1 h. The method is therefore recommended as a screening tool on farms for the rapid diagnosis of TiLV.

Here, we present the complete coding sequences of two tilapia lake virus (TiLV) isolates recovered during an investigation of a mortality event in farmed Nile tilapia in the United States. Phylogenetic analysis supported the isolates as each other's closest relatives and members of a clade of Thai TiLV strains., (Copyright © 2020 Ahasan et al.)

Tilapia lake virus (TiLV) is an emerging virus associated with high fish mortality and economic losses. This study investigates the virucidal effects of the following disinfectants (active ingredients) on TiLV: 2.5 ppm iodine, 10 ppm sodium hypochlorite (NaOCl), 300 ppm hydrogen peroxide (H 2 O 2 ), 80 ppm formalin and 5,000 ppm (0.5%) Virkon ® . Factors that affect the disinfectants' efficacy, including temperature, contact time and soiling (organic matter) interference, were examined under conditions mimicking natural aquaculture practices. TiLV inactivation of higher than 5 log 10 TCID 50  ml -1 was achieved after 10 min and at 28°C for all disinfectants except formalin; similar inactivation levels were reached by NaOCl and Virkon ® at 10 min and 4°C. Extended exposure to formalin from 10 to 60 min at 28°C rendered more than 5 log 10 inactivation. Increasing synthetic organic matter in the water to mimic soiling interference reduced the efficacy of NaOCl, iodine and H 2 O 2 when tested at 10 min and 28°C; however, Virkon ® still achieved more than 5 log 10 inactivation. This study demonstrates that most common disinfectants effectively reduced viral loads to minimum levels. To limit the spread of TiLV in aquaculture farms and related facilities, the appropriate use of such disinfectants should therefore be promoted and implemented., (© 2019 John Wiley & Sons Ltd.)

To gain a better understanding of the pathogenesis of tilapia lake virus (TiLV) infections in Nile tilapia (Oreochromis niloticus), fingerlings were challenged with a single dose of 1 × 10 4  TCID 50 /fish of TiLV utilizing intracoelomic/intraperitoneal (IC ch ) or intragastric (IG ch ) routes. Acute mortalities were present in both groups, reaching 70 and 40% in IC ch and IG ch after 10 days, respectively. Challenged fish presented erratic swimming, lethargy, anorexia, exophthalmia and cutaneous petechiae and ecchymoses. Histological changes in challenged groups included syncytial formation, intracytoplasmic inclusion bodies and multifocal hepatocellular degeneration and necrosis. In addition, multifocal areas of mild proliferation of glial cells and lymphocytic perivascular cuffing were observed in the brain of exposed challenged groups. TiLV RNA was detected in gills and faeces of challenged fish using quantitative reverse transcriptase-PCR, as well as in the tank water holding challenged fish. Moreover, TiLV RNA was detected in scrolls obtained from formalin-fixed paraffin-embedded tissue blocks from challenged fish. Results from this study suggest that IG methods represent an additional method to study the pathogenesis of the disease in this species, as it results in infection and diseases as in naturally occurring cases and does not bypass important mucosal immune responses as injectable routes do., (© 2019 John Wiley & Sons Ltd.)

Tilapia lake virus disease (TiLVD) is an emerging viral disease in tilapia with worldwide distribution. Although the horizontal transmission of TiLV has been demonstrated through the cohabitation of infected fish with susceptible fish, no direct experiment showed the potential of vertical transmission from broodstock to progeny. In this study, natural outbreaks of TiLV in broodstock and fry in two tilapia hatcheries were confirmed. The TiLV genomic RNA was detected in liver and reproductive organs of infected broodstock, while infective virus was isolated in susceptible cell line. In situ hybridization assay confirmed the presence of TiLV in the ovary and testis of naturally infected fish and experimentally challenged fish. Moreover, early detection of TiLV in 2-day-old fry and the presence of TiLV genomic RNA and viable virus in the testis and ovary suggested the possible transfer of this virus from infected broodstock to progenies. As infective virus was present in gonads and fry in natural outbreak and experimental fish, the importance of biosecurity and prevention of the virus to establish in the hatchery should be emphasized. Hence, the development of TiLV-free broodstock and the maintenance of high biosecurity standards in the hatcheries are essential for any attempt of virus eradication., (© 2019 John Wiley & Sons Ltd.)

Recent outbreaks of a novel tilapia lake virus (TiLV) have raised concerns regarding the international spread of TiLV in frozen tilapia products. This study investigated the potential risks of frozen tilapia fillet as a source of TiLV transmission. It revealed that TiLV genomic RNA could be detected in tilapia fillet and the virus isolated from non-frozen and frozen fillets with clinical TiLV infection stored up to 28 days caused a cytopathic effect (CPE) formation in the susceptible cell line in vitro. However, frozen fillets from clinical TiLV infection stored for 90 and 120 days did not cause CPE in the susceptible cell line. Similarly, CPE was not observed in TiLV isolated from subclinically TiLV-infected fish fillets. In addition, in vivo bioassay revealed that despite the presence of TiLV isolated from subclinically TiLV-infected fillet stored at -20°C for 14 days, there was no evidence of TiLV disease in naïve red hybrid tilapia based on the absence of clinical signs and mortality and without the detection of TiLV genomic RNA using reverse transcription-quantitative polymerase chain reaction assay. Collectively, these findings suggested minimal risk of transmission of TiLV via frozen tilapia fillets., (© 2018 John Wiley & Sons Ltd.)

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be used as part of surveillance programs, biosecurity measures and in TiLV basic research laboratories. The gold standard of virus diagnostics typically involves virus isolation followed by complementary techniques such as reverse-transcription polymerase chain reaction (RT-PCR) for further verification. This can be cumbersome, time-consuming and typically requires tissue samples heavily infected with virus. The use of RT-quantitative (q)PCR in the detection of viruses is advantageous because of its quantitative nature, high sensitivity, specificity, scalability and its rapid time to result. Here, the entire method of PCR based approaches for TiLV detection is described, from tilapia organ sectioning, total ribonucleic acid (RNA) extraction using a guanidium thiocyanate-phenol-chloroform solution, RNA quantification, followed by a two-step PCR protocol entailing, complementary deoxyribonucleic acid (cDNA) synthesis and detection of TiLV by either conventional PCR or quantitative identification via qPCR using SYBR green I dye. Conventional PCR requires post-PCR steps and will simply inform about the presence of the virus. The latter approach will allow for absolute quantification of TiLV down to as little as 2 copies and thus is exceptionally useful for TiLV diagnosis in sub-clinical cases. A detailed description of the two PCR approaches, representative results from two laboratories and a thorough discussion of the critical parameters of both have been included to ensure that researchers and diagnosticians find their most suitable and applicable method of TiLV detection.

Since 2015, a novel orthomyxo-like virus, tilapia lake virus (TiLV) has been associated with outbreaks of disease and massive mortality of cultured Nile and red tilapia (Oreochromis niloticus and Oreochromis spp., respectively) in Thailand. In this study, TiLV was isolated from field samples and propagated in the permissive E-11 cell line, with cytopathic effect (CPE) development within 3-5days post-inoculation. Electron micrographs of infected E-11 cells and fish tissues confirmed the rounded, enveloped virions of 60 to 80nm with characteristics very similar to those of Orthomyxoviridae. In vivo challenge studies showed that high mortality in Nile (86%) and red tilapia (66%) occurred within 4-12days post-infection. The virus was re-isolated from challenged fish tissues in the permissive cell line, and PCR analysis confirmed TiLV as a causative pathogen. The distinct histopathology of challenged fish included massive degeneration and inflammatory cell infiltration in the liver and brain as well as the presence of eosinophilic intracytoplasmic inclusions in hepatocytes and splenic cells. Our results fulfilled Koch's postulates and confirmed that TiLV is an etiologic agent of mass mortality of tilapia in Thailand. The emergence of this virus in many countries has helped increase awareness that it is a potential threat to tilapia aquacultured in Thailand, Asia, and worldwide., (Copyright © 2017 Elsevier B.V. All rights reserved.)