Urgent update on possible worldwide spread of tilapia lake virus (TiLV)

Tilapia lake virus disease (TiLVD) (also known as syncytial hepatitis of tilapia-SHT) is a newly emerging viral disease of tilapia caused by tilapia lake virus (TiLV), a novel Orthomyxo-like virus (Ferguson et al. 2014; Eyngor et al. 2014; Bacharach et al. 2016; Del-Pozo et al. 2017; NACA, 2017; OIE, 2017). Occurrence of the disease was officially documented earliest in Ecuador and Israel in 2013 and 2014, respectively (Ferguson et al. 2014; Eyngor et al. 2014). The virus, however, is believed to have been responsible for massive mortalities in farmed tilapia in Israel since 2009 (Eyngor et al. 2014). Infection of TiLV was later reported from Colombia (Kembou Tsofack et al. 2017) and Egypt (Fathi et al. 2017) and most recently from Thailand (Dong et al. 2017a; Surachetpong et al. 2017). Natural disease outbreaks result in variable mortalities ranging from 9.2 to 90%, with tilapia fingerlings and juveniles being more vulnerable than larger fish (Ferguson et al. 2014; Fathi et al. 2017; Dong et al. 2017a; Surachetpong et al. 2017). Unlike other viral diseases of tilapia, TiLV appears to be widely spread and so may be present in many countries where it is not yet recognized.

Recently, we released a warning of TiLV in Thailand, including an improved semi-nested RT-PCR method for rapid detection and we urge those involved in Tilapia culture to test for the virus in their country (Dong et al. 2017b). The Fish Health Platform in Centex, BIOTEC/Mahidol University) has also obtained positive test results for TiLV from other countries in Asia where it has not yet been reported, supporting our appeal for wider testing. Further, the majority of our archived samples collected from previous disease outbreaks in several tilapia hatcheries in Thailand during 2012-2017 have tested positive for TiLV (unpublished data), indicating the presence of TiLV in Thailand even before the virus became known to science in 2013. The origin of the disease is currently unknown, but many countries have been translocating tilapia fry/fingerlings prior to and even after the description of TiLV.  Based on records we could obtain about such translocations, we have prepared a map that contains a list of 5 countries with confirmed reports of TiLV infections (red, Fig.1) and a list of 43 other countries that we believe have imported tilapia that may have been infected with TiLV (blue, Fig. 1). We hope that widespread surveillance for TiLV in the Tilapia industry and in translocated fish will help reduce the impact and spread of this disease. To this end, we repeat our willingness to provide on request a free positive control plasmid for use with the method we have developed to detect TiLV by semi-nested PCR (Dong et al. 2017b).

600 dpi

Fig. 1 Possible worldwide distribution map of TiLV. Countries with confirmatory evidence of TiLV circulation (red colour). Blue colour indicates 43 countries that we believe have imported tilapia that may have been infected with TiLV.

List of countries with confirmed evidence of TiLV: Ecuador, Israel, Colombia, Egypt and Thailand

List of countries we believe at high risk of TiLV: Algeria, Bahrain, Bangladesh, Belgium, Burundi, Canada, China, Congo, ElSalvador, Germany, Guatemala, India, Indonesia, Japan, Jordan, Laos, Malaysia, Mexico, Mozambique, Myanmar, Nepal, Nigeria, Pakistan, Philippines, Romania, Rwanda, Saudi Arabia, Singapore, South Africa, Sri Lanka, Switzerland, Tanzania, Togo, Tunisia, Turkey, Turkmenistan, Uganda, Ukraine, United Arab Emirate, United Kingdom, United States, Vietnam and Zambia.


  • We recommend that the 43 countries we have listed quickly initiate surveillance for TiLV in cultured Tilapia, since the virus may have been introduced via direct or indirect translocation of fry/fingerlings from the 5 countries where it has been reported.
  • Biosecurity should be applied to prevent wider spread of the disease especially by countries with no predictive record of TiLV risk.
  • Since TiLV infects very early developmental stages of tilapia (fertilized eggs, fry, and fingerlings) when fish immune system is not fully developed, the use of vaccines may not be an effective control approach.
  • Research should be promoted for the development of methods to clear TiLV from infected tilapia broodstock and allow production TiLV-free fry/fingerlings.
  • Programs should be promoted to develop Tilapia stocks specific pathogen free (SPF) for TiLV and other pathogens as a potential approach to limit impact of Tilapia diseases globally.
  • Since TiLV infections result in highly variable mortality (9.2-90%), it is urgent that research should be promoted to discover the underlying reasons (e.g., research on the correlation between TiLV virulence and genetic types or other factors).


The authors acknowledge fish producers who provided fish samples and information for this study. We would also like to thank Prof. T.W. Flegel for his advice and assistance in preparing this announcement.


  1. Bacharach, E., Mishra, N., Briese, T., Zody, M.C., Kembou Tsofack, J.E., Zamostiano, R., Berkowitz, A., Ng, J., Nitido, A., Corvelo, A., Toussaint, N.C., Abel Nielsen, S.C., Hornig, M., Del Pozo, J., Bloom, T., Ferguson, H., Eldar, A., Lipkin, W.I., 2016. Characterization of a novel Orthomyxo-like virus causing cass die-offs of tilapia. MBio. 7, e00431-00416.
  2. Del-Pozo, J., Mishra, N., Kabuusu, R., Cheetham, S., Eldar, A., Bacharach, E., Lipkin, W.I., Ferguson, H.W., 2017. Syncytial Hepatitis of Tilapia (Oreochromis niloticus) is associated with Orthomyxovirus-Like virions in hepatocytes. Vet Pathol. 54, 164-170.
  3. Dong, H.T., Siriroob, S., Meemetta, W., Santimanawong, W., Gangnonngiw, W., Pirarat, N., Khunrae, K., Rattanarojpong, T., Vanichviriyakit, R., Senapin, S., 2017a. Emergence of tilapia lake virus in Thailand and an alternative semi-nested RT-PCR for detection. Aquaculture. 476, 111-118.
  4. Dong HT, Siriroob, S., Meemetta, W., Santimanawong, W., Gangnonngiw, W., Pirarat, N., Khunrae, P., Rattanarojpong, T., Vanichviriyakit, R. and Senapin, S., 2017b. A warning and an improved PCR detection method for tilapia lake virus (TiLV) disease in Thai tilapia farms. https://enaca.org/?id=858
  5. Eyngor, M., Zamostiano, R., Kembou Tsofack, J.E., Berkowitz, A., Bercovier, H., Tinman, S., Lev, M., Hurvitz, A., Galeotti, M., Bacharach, E., Eldar, A., 2014. Identification of a novel RNA virus lethal to tilapia. J Clin Microbiol. 52, 4137-4146.
  6. Fathi, M., Dickson, C., Dickson, M., Leschen, W., Baily, J., Fiona, M., Ulrich, K., Weidmann, M., 2017. Identification of Tilapia Lake Virus in Egypt in Nile tilapia affected by ‘summer mortality’ syndrome. Aquaculture. 473, 430–432.
  7. Ferguson, H.W., Kabuusu, R., Beltran, S., Reyes, E., Lince, J.A., del Pozo, J., 2014. Syncytial hepatitis of farmed tilapia, Oreochromis niloticus (L.): a case report. J Fish Dis. 37, 583-589.
  8. NACA (Network of Aquaculture Centres in Asia-Pacific), 2017. Tilapia Lake Virus (TiLV) – an Emerging Threat to Farmed Tilapia in the Asia-Pacific Region. Disease Advisory. https://enaca.org/?id=864&title=tilapia-lake-virus-disease-advisory
  9. OIE (World Organisation For Animal Health), 2017. Tilapia lake virus (TiLV)-A novel Orthomyxo-like virus. May, 2017. http://www.oie.int/en/international-standard-setting/specialists-commissions-groups/aquatic-animal-commission-reports/disease-information-cards/
  10. Surachetpong, W., Janetanakit, T., Nonthabenjawan, N., Tattiyapong, P., Sirikanchana, K., Amonsin, A., Outbreaks of tilapia lake virus infection, Thailand, 2015-2016. Emerg Infect Dis. https://dx.doi.org/10.3201/eid2306.161278
  11. Kembou Tsofack, J.E., Zamostiano, R., Watted, S., Berkowitz, A., Rosenbluth, E., Mishra, N., Briese, T., Lipkin, W.I., Kabuusu, R.M., Ferguson, H., Del Pozo, J., Eldar, A., Bacharach, E., 2017. Detection of tilapia lake virus in clinical samples by culturing and nested reverse transcription-PCR. J Clin Microbiol. 55, 759-767.

Source: https://enaca.org/?id=870&title=urgent-update-on-possible-worldwide-spread-of-tilapia-lake-virus-tilv


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A warning and an improved PCR detection method for tilapia lake virus (TiLV) disease in Thai tilapia farms

H.T. Dong, S. Siriroob, W. Meemetta, W. Santimanawong, W. Gangnonngiw, N. Pirarat, P. Khunrae, T. Rattanarojpong, R. Vanichviriyakit and S. Senapin

Tilapia lake virus (TiLV) is a novel RNA virus resembling Orthomyxovirus. It has been reported as a newly emerging virus that causes syncytial hepatitis of tilapia (SHT) in Israel, Ecuador, Colombia, and Egypt (Ferguson et al. 2014; Eyngor et al. 2014; Bacharach et al. 2016; Del-Pozo et al. 2017, Fathi et al. 2017). The disease caused massive mortality up to 90% in farmed tilapia and is considered to be a potential threat to global tilapia farming (Eyngor et al. 2014).

In Thai tilapia farms, disease outbreaks have recently occurred associated with high cumulative mortalities (20-90%), and we have found that the diseased fish show typical histopathological features of SHT. Infection has been confirmed by transmission electron microscopy (TEM), in situ hybridization and high nucleotide sequence identity to TiLV from Israel (Dong et al. 2017). Our research progress was delayed by use of nested RT-PCR primer sequences previously published by Eyngor et al. (2014) together with detailed RT-PCR protocols later released by Tsofack et al. (2016). Our preliminary detection assays performed using the earlier report (Eyngor et al. 2014) with assumed PCR conditions (not specified in the publication) resulted in amplification of nonspecific fish genes. This probably resulted from the fact that 13-18 out of 20 nucleotides for the primer “Nested ext-2” matched fish genes. We, therefore, modified the nested RT-PCR protocols into a semi-nested RT-PCR by omitting the primer “Nested ext-2” to avoid false positive results.

The semi-nested RT-PCR protocol given below may be used freely for non-commercial applications to detect TiLV. Heavily-infected samples will generate 2 amplicon bands of 415 bp and 250 bp while lightly-infected samples will generate a single 250-bp amplicon band (Fig. 1). Since this is the first report of TiLV in Southeast Asia, the authors urge fish health laboratories in Asia to test for TiLV when abnormal mortality of tilapia occurs. Please contact Centex Shrimp (saengchan ‘at’ biotec.or.th) to obtain positive control plasmids (pGEM-415_bp), which will be provided free for non-commercial organisations, and for a fee to the private sector.

PCR gel

Fig. 1 A sample agarose gel of TiLV detection from fish RNA extracts. Expected band sizes of 415 bp and 250 bp represent amplicons from the first and semi-nested PCR, respectively, with lanes marked ++ for a heavy infection, + for a light infection and – for the negative control. The band marked with an asterisk (*) on the right side of the gel probably arose from cross hybridization of the amplified products. M = DNA marker

Source: https://enaca.org/?id=858

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Emergence of tilapia lake virus in Thailand and an alternative semi-nested RT-PCR for detection


  • Outbreaks of TiLV associated with massive mortalities in farmed tilapia in Thailand.
  • An alternative semi-nested RT-PCR has been developed for disease diagnosis.
  • In situ hybridization assay revealed multiple tissues tropism of the virus.
  • Transmission electron microscopy revealed intracytoplasmic viral particles.
  • Partial genome of TiLV from Thailand exhibited genetic variations.

TiLV-Histopathology HT Dong

Histopathological feature of syncytial hepatitis of tilapia (SHT) observed from the liver of  TiLV-infected tilapia (Photo: HT Dong)


The present study reports outbreaks of tilapia lake virus (TiLV), an emerging pathogen causing syncytial hepatitis of tilapia (SHT), in farmed tilapia in Thailand. Occurrence of the virus was confirmed by RT-PCR and nucleotide sequence homology to the TiLV from Israel. Diseased fish exhibited typical histopathological features of syncytial giant cells in the liver examined through H&E and semi-thin sections. Presence of intracytoplasmic viral particles was revealed by TEM. In situ hybridization using a specific DIG-labeled probe derived from a partial genome segment 3 of TiLV genome revealed multiple tissues tropism of the virus including liver, kidney, brain, spleen, gills and connective tissue of muscle. An alternative semi-nested RT-PCR protocol has been developed in this study for disease diagnosis. Additionally, comparative genetic analysis revealed genetic variations of Thailand-originated virus to the Israel TiLV strains, sharing 96.28 to 97.52% nucleotide identity and 97.35 to 98.84% amino acid identity. Outbreaks of TiLV in different continents might signal a serious threat to tilapia aquaculture globally.

Source: http://www.sciencedirect.com/science/article/pii/S0044848617303484


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Aeromonas jandaei and Aeromonas veronii caused disease and mortality in Nile tilapia, Oreochromis niloticus (L.)

Dong et al. 2017 – Journal of Fish Diseases. doi:10.1111/jfd.12617



Diseases caused by motile aeromonads in freshwater fish have been generally assumed to be linked with mainly Aeromonas hydrophila while other species were probably overlooked. Here, we identified two isolates of non-A. hydrophila recovered from Nile tilapia exhibiting disease and mortality after exposed to transport-induced stress and subsequently confirmed their virulence in artificial infection. The bacterial isolates were identified as Aeromonas jandaei and Aeromonas veronii based on phenotypic features and homology of 16S rDNA. Experimental infection revealed that the high dose of A. jandaei (3.7 × 106 CFU fish−1) and A. veronii (8.9 × 106 CFU fish−1) killed 100% of experimental fish within 24 h, while a 10-fold reduction dose killed 70% and 50% of fish, respectively. When the challenge dose was reduced 100-fold, mortality of the fish exposed to A. jandaei and A. veronii decreased to 20% and 10%, respectively. The survivors from the latter dose administration were rechallenged with respective bacterial species. Lower mortality of rechallenged fish (0%–12.5%) compared to the control groups receiving a primary infection (37.5%) suggested that the survivors after primary infection were able to resist secondary infection. Fish exposed to either A. jandaei or A. veronii exhibited similar clinical signs and histological manifestation.

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Recovery of Vibrio harveyi from scale drop and muscle necrosis disease in farmed barramundi, Lates calcarifer in Vietnam


  • Emergence of scale drop and muscle necrosis disease in farmed barramundi in Vietnam
  • Coinfections of culturable and unculturable bacteria were uncovered from diseased fish.
  • A pathogenic V. harveyi strain was identified as the main causative agent.
  • Fish infected with V. harveyi exhibited similar clinical signs and unique histological changes of naturally diseased fish.
  • The role of unculturable bacteria needs further investigation.



Symptoms of scale drop and muscle necrosis have been considered as an emerging problem in farmed barramundi (Lates calcarifer) in Vietnam since 2013. Naturally diseased fish exhibited remarkable external clinical signs of scale loss, muscle degradation and eventually died. The objective of this study was to determine the infectious causative agent of the clinically diseased fish collected from barramundi caged culture in central Vietnam in 2015. Histological examination from naturally sick fish revealed signs of severe necrotic muscles with infiltration of massive immune-related cells, severe hemorrhage and blood congestion in the brain, collapsed kidney tubules and epithelial cells sloughing into the lumen. Five different bacterial species were recovered from diseased fish and putatively identified as Vibrio harveyi, Vibrio tubiashii, Tenacibaculum litopenaei, Tenacibaculum sp. and Cytophaga sp. based on homology of 16S rDNA sequences and biochemical characteristics. Experimental infection revealed that only V. harveyi killed the fish with similar clinical signs and histological changes compared to naturally diseased fish. Additionally, several unculturable bacteria including T. maritimum were also uncovered from DNA extracted from necrotic muscles by species-specific PCR and 16S rDNA clone library sequencing, but their roles in disease manifestation need further investigation.


Barramundi; Scale drop; Muscle necrosis; Vibrio harveyi; Unculturable bacteria

Source: http://www.sciencedirect.com/science/article/pii/S0044848616309000

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Outbreaks of ulcerative disease associated with ranavirus infection in barcoo grunter, Scortum barcoo (McCulloch & Waite)

How to cite: Kayansamruaj, P., Rangsichol, A., Dong, H. T., Rodkhum, C., Maita, M., Katagiri, T. and Pirarat, N. (2017), Outbreaks of ulcerative disease associated with ranavirus infection in barcoo grunter, Scortum barcoo (McCulloch & Waite). J Fish Dis. doi:10.1111/jfd.12606


In 2013, an outbreak of ulcerative disease associated with ranavirus infection occurred in barcoo grunter, Scortum barcoo (McCulloch & Waite), farms in Thailand. Affected fish exhibited extensive haemorrhage and ulceration on skin and muscle. Microscopically, the widespread haemorrhagic ulceration and necrosis were noted in gill, spleen and kidney with the presence of intracytoplasmic eosinophilic inclusion bodies. When healthy barcoo grunter were experimentally challenged via intraperitoneal and oral modes with homogenized tissue of naturally infected fish, gross and microscopic lesions occurred with a cumulative mortality of 70–90%. Both naturally and experimentally infected fish yielded positive results to the ranavirus-specific PCR. The full-length nucleotide sequences of major capsid protein gene of ranaviral isolates were similar to largemouth bass virus (LMBV) and identical to largemouth bass ulcerative syndrome virus (LBUSV), previously reported in farmed largemouth bass (Micropterus salmoides L.), which also produced lethal ulcerative skin lesions. To the best of our knowledge, this is the first report of a LMBV-like infection associated with skin lesions in barcoo grunter, adding to the known examples of ranavirus infection associated with skin ulceration in fish.

Source: http://onlinelibrary.wiley.com/doi/10.1111/jfd.12606/full

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Severe clinical signs of diseased tilapia infected by Streptococcus agalactiae


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