Development of quantitative real-time PCR (qPCR) assays for Tilapinevirus tilapiae and progression of infection in experimentally infected Nile tilapia

A Jancy Robina; B. Madhusudhana Rao; Anutosh Paria; A. Deepika; Sagar K. Hundare; M. G. Pratapa; Megha K. Bedekar; Saurav Kumar; Neeraj Sood; Pravata Kumar Pradhan; K. V. Rajendran

doi:10.21077/ijf.2026.73.3.181724-12

Authors

A Jancy Robina
B. Madhusudhana Rao
Anutosh Paria
A. Deepika
Sagar K. Hundare
M. G. Pratapa
Megha K. Bedekar
Saurav Kumar
Neeraj Sood
Pravata Kumar Pradhan NBFGR, Lucknow
K. V. Rajendran

https://doi.org/10.21077/ijf.2026.73.3.181724-12

Keywords:

Tilapinevirus tilapiae, Tilapia lake virus, TiLV, qPCR

Abstract

Tilapia lake virus disease caused by Tilapia lake virus (TiLV) or Tilapinevirus tilapiae is a highly infectious disease of farmed tilapia. In this study, two SYBR Green-based quantitative real-time PCR (qPCR) assays targeting the conserved sequences of genomic segment 10 and 1 of TiLV were developed and the assays were applied to track the progression of virus replication in experimentally infected tilapia (Oreochromis niloticus). The qPCR assays showed a wide dynamic range of detection with the reliable limit of detection (LOD) of 10 and 100 copies for segment 10 and 1, respectively. Further, the PCR efficiency (mean±SD) estimated in 5 different experiments for segment 10 and 1 were 97.05%±3.35 and 101.41%±3.63, respectively. qPCR targeting segment 10 showed high reproducibility within and between the assays. Inter and intra-assay variation ranged from 0.06 to 1.55% and 0.01 to 6.05%, respectively. The assays developed were applied to quantify TiLV load in different target tissues viz. liver, brain, spleen, and mucus of experimentally infected tilapia and Nile tilapia heart (OnH) cell line. Among the tissues, the liver showed the highest viral load (3.2x106 to 1.0x108 viral copies 100 ng-1 RNA), and mucus had the lowest viral load (3.0x103 to 1.0x105 viral copies 100 ng-1 RNA). Further, comparison of viral loads estimated using assays targeting genomic segments 10 and 1 in liver tissue revealed that segment 10-specific qPCR assay consistently detected higher TiLV copy numbers, at all post-infection time points. The specificity of both qPCR assays was also tested against Betanodavirus infecting sea bass. Based on these findings, the newly developed assay targeting genomic segment 10 can be recommended as a reliable diagnostic tool for TiLV detection. Liver tissue is proposed as the most suitable target tissue for TiLV detection, while mucus as a non-invasive sample for detection.

Keywords: Betanodavirus, Farmed tilapia, Oreochromis niloticus, SYBR Green, Tilapia lake virus, TiLV

Downloads

Download data is not yet available.

Author Biography

Pravata Kumar Pradhan, NBFGR, Lucknow

NBFGR, Lucknow

References

1. Abdullah, A., Ramly, R., Ridzwan, M.M., Sudirwan, F., Abas, A., Ahmad, K., Murni, M. and Kua, B.C. 2018. First detection of tilapia lake virus (TiLV) in wild river carp (Barbonymus schwanenfeldii) at Timah Tasoh Lake, Malaysia. J. Fish Dis., 41(9):1459-1462. https://doi.org/10.1111/jfd.12843. DOI: https://doi.org/10.1111/jfd.12843

2. Ahasan, M.S., Keleher, W., Giray, C., Perry, B., Surachetpong, W., Nicholson, P., Al-Hussinee, L., Subramaniam, K. and Waltzek, T.B. 2020. Genomic characterization of tilapia lake virus isolates recovered from moribund Nile tilapia (Oreochromis niloticus) on a farm in the United States. Microbiol. Resour. Announc., 9(4):e01368-19. https://doi.org/10.1128/mra.01368-19. DOI: https://doi.org/10.1128/MRA.01368-19

3. Amal, M.N.A., Koh, C.B., Nurliyana, M., Suhaiba, M., Nor-Amalina, Z., Santha, S., Diyana-Nadhirah, K.P., Yusof, M.T., Ina-Salwany, M.Y. and Zamri-Saad, M. 2018. A case of natural co-infection of Tilapia Lake Virus and Aeromonas veronii in a Malaysian red hybrid tilapia (Oreochromis niloticus× O. mossambicus) farm experiencing high mortality. Aquaculture, 485:12-16. https://doi.org/10.1016/j.aquaculture.2017.11.019. DOI: https://doi.org/10.1016/j.aquaculture.2017.11.019

4. Bacharach, E., Mishra, N., Briese, T., Zody, M.C., Kembou Tsofack, J.E., Zamostiano, R., Berkowitz, A., Ng, J., Nitido, A., Corvelo, A. and Toussaint, N.C. 2016. Characterization of a novel orthomyxo-like virus causing mass die-offs of tilapia. MBio, 7(2):e00431-16. https://doi.org/10.1128/mbio.00431-16. DOI: https://doi.org/10.1128/mBio.00431-16

5. Behera, B.K., Pradhan, P.K., Swaminathan, T.R., Sood, N., Paria, P., Das, A., Verma, D.K., Kumar, R., Yadav, M.K., Dev, A.K. and Parida, P.K. 2018. Emergence of tilapia lake virus associated with mortalities of farmed Nile tilapia Oreochromis niloticus (Linnaeus 1758) in India. Aquaculture, 484:168-174. https://doi.org/10.1016/j.aquaculture.2017.11.025. DOI: https://doi.org/10.1016/j.aquaculture.2017.11.025

6. Bustin, S.A. 2000. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol., 25(2):169-93. https://doi.org/10.1677/jme.0.0250169. DOI: https://doi.org/10.1677/jme.0.0250169

7. Chaput, D.L., Bass, D., Alam, M., Al Hasan, N., Stentiford, G.D., Van Aerle, R., Moore, K., Bignell, J.P., Haque, M.M. and Tyler, C.R. 2020. The segment matters: Probable reassortment of tilapia lake virus (TiLV) complicates phylogenetic analysis and inference of geographical origin of new isolate from Bangladesh. Viruses, 12(3):258. https://doi.org/10.3390/v12030258. DOI: https://doi.org/10.3390/v12030258

8. Chengula, A.A., Mugimba, K.K., Tal, S., Levi, R.T., Dubey, S., Mutoloki, S., Dishon, A., David, L., Evensen, Ø. and Munang’andu, H.M. 2022. Efficiency, sensitivity and specificity of a quantitative real-time PCR assay for Tilapia Lake virus (TiLV). J. Virol. Methods, 307:114567. https://doi.org/10.1016/j.jviromet.2022.114567. DOI: https://doi.org/10.1016/j.jviromet.2022.114567

9. Contreras, H., Vallejo, A., Mattar, S., Ruiz, L., Guzmán, C. and Calderón, A. 2021. First report of tilapia lake virus emergence in fish farms in the department of Córdoba. Colombia Vet. World, 14(4):865-872. www.doi.org/10.14202/vetworld.2021.865-872. DOI: https://doi.org/10.14202/vetworld.2021.865-872

10. Debnath, P.P., Delamare‐Deboutteville, J., Jansen, M.D., Phiwsaiya, K., Dalia, A., Hasan, M.A., Senapin, S., Mohan, C.V., Dong, H.T. and Rodkhum, C. 2020. Two‐year surveillance of tilapia lake virus (TiLV) reveals its wide circulation in tilapia farms and hatcheries from multiple districts of Bangladesh. J. Fish Dis., 43(11):1381-1389. https://doi.org/10.1111/jfd.13235. DOI: https://doi.org/10.1111/jfd.13235

11. Del-Pozo, J., Mishra, N., Kabuusu, R., Cheetham, S., Eldar, A., Bacharach, E., Lipkin, W.I. and Ferguson, H.W. 2017. Syncytial hepatitis of tilapia (Oreochromis niloticus) is associated with orthomyxovirus-like virions in hepatocytes. Vet. Pathol., 54(1):164-170. https://doi.org/10.1177/0300985816658100. DOI: https://doi.org/10.1177/0300985816658100

12. Dong, H.T., Siriroob, S., Meemetta, W., Santimanawong, W., Gangnonngiw, W., Pirarat, N., Khunrae, P., Rattanarojpong, T., Vanichviriyakit, R. and Senapin, S. 2017. Emergence of tilapia lake virus in Thailand and an alternative semi-nested RT-PCR for detection. Aquaculture, 476:111-118. https://doi.org/10.1016/j.aquaculture.2017.04.019. DOI: https://doi.org/10.1016/j.aquaculture.2017.04.019

13. Eyngor, M., Zamostiano, R., Kembou Tsofack, J.E., Berkowitz, A., Bercovier, H., Tinman, S., Lev, M., Hurvitz, A., Galeotti, M., Bacharach, E. and Eldar, A. 2014. Identification of a novel RNA virus lethal to tilapia. J. Clin. Microbiol., 52(12):4137-4146. https://doi.org/10.1128/jcm.00827-14. DOI: https://doi.org/10.1128/JCM.00827-14

14. Fathi, M., Dickson, C., Dickson, M., Leschen, W., Baily, J., Muir, F., Ulrich, K. and Weidmann, M. 2017. Identification of Tilapia Lake Virus in Egypt in Nile tilapia affected by ‘summer mortality’ syndrome. Aquaculture, 473:430-432. https://doi.org/10.1016/j.aquaculture.2017.03.014. DOI: https://doi.org/10.1016/j.aquaculture.2017.03.014

15. Ferguson, H.W., Kabuusu, R., Beltran, S., Reyes, E., Lince, J.A. and del Pozo, J. 2014. Syncytial hepatitis of farmed tilapia, Oreochromis niloticus (L.): a case report. J. Fish Dis., 37(6):583-589. https://doi.org/10.1111/jfd.12142. DOI: https://doi.org/10.1111/jfd.12142

16. He, T., Zhang, Y-Z., Gao, L-J., Miao, B., Zheng, J-S., Pu, D-C., Zhang, Q-Q., Zeng, W-W., Wang, D-S., Su, S-Q. and Zhu, S. 2023. Identification and pathogenetic study of tilapia lake virus (TiLV) isolated from naturally diseased tilapia. Aquaculture, 565:739166. https://doi.org/10.1016/j.aquaculture.2022.739166. DOI: https://doi.org/10.1016/j.aquaculture.2022.739166

17. ICTV, 2022. International Committee on Taxonomy of Viruses. https://ictv.global/taxonomy

18. Kampeera, J., Dangtip, S., Suvannakad, R., Khumwan, P., Senapin, S. and Kiatpathomchai, W. 2021. Reverse transcription loop‐mediated isothermal amplification (RT‐LAMP) combined with colorimetric gold nanoparticle (AuNP) probe assay for visual detection of tilapia lake virus (TiLV) in Nile and red hybrid tilapia. J. Fish Dis., 44(10):1595-1607. https://doi.org/10.1111/jfd.13482. DOI: https://doi.org/10.1111/jfd.13482

19. Liamnimitr, P., Thammatorn, W., Sonicha, U., Tattiyapong, P. and Surachetpong, W. 2018. Non-lethal sampling for Tilapia Lake Virus detection by RT-qPCR and cell culture. Aquaculture, 486:75-80. https://doi.org/10.1016/j.aquaculture.2017.12.015. DOI: https://doi.org/10.1016/j.aquaculture.2017.12.015

20. MacKay, I.M. 2004. Real-time PCR in the microbiology laboratory. Clin. Microbiol. Infect., 10(3):190-212. https://doi.org/10.1111/j.1198-743X.2004.00722.x. DOI: https://doi.org/10.1111/j.1198-743X.2004.00722.x

21. Megarani, D.V., Al-Hussinee, L., Subramaniam, K., Sriwanayos, P., Imnoi1, K., Keleher, B., Nicholson, P., Surachetpong, W., Tattiyapong, P., Hick, P., Gustafson, L.L. and Waltzek, T.B. 2022. Development of a TaqMan quantitative reverse transcription PCR assay to detect tilapia lake virus. Dis. Aquat. Org., 152:147-158. https://doi.org/10.3354/dao03700. DOI: https://doi.org/10.3354/dao03700

22. Mugimba, K.K., Chengula, A.A., Wamala, S., Mwega, E.D., Kasanga, C.J., Byarugaba, D.K., Mdegela, R.H., Tal, S., Bornstein, B., Dishon, A., Mutoloki, S., David, L., Evensen, Ø. and Munang'andu, H.M. 2018. Detection of Tilapia lake virus (TiLV) infection by PCR in farmed and wild Nile tilapia (Oreochromis niloticus) from Lake Victoria. J. Fish Dis., 41(8):1181-1189. https://doi.org/10.1111/jfd.12790. DOI: https://doi.org/10.1111/jfd.12790

23. Nicholson, P., Fathi, M.A., Fischer, A., Mohan, C., Schieck, E., Mishra, N., Heinimann, A., Frey, J., Wieland, B. and Jores, J. 2017. Detection of Tilapia Lake Virus in Egyptian fish farms experiencing high mortalities in 2015. J. Fish Dis., 40:1925-1928. https://doi.org/10.1111/jfd.12650. DOI: https://doi.org/10.1111/jfd.12650

24. Nicholson, P., Rawiwan, P. and Surachetpong, W. 2018. Detection of Tilapia lake virus using conventional RT-PCR and SYBR Green RT-qPCR. J. Vis. Exp., 141:e58596. http://dx.doi.org/10.3791/58596. DOI: https://doi.org/10.3791/58596-v

25. OIE, 2017a. Immediate notifications and follow-ups. Tilapia lake virus, Chinese Taipei. Retrieved from https://www.oie.int/wahis_2/publi c/wahid.php/Revie wrepo rt/Revie w?repor tid=24033.rt/Revie w?repor tid=24033.

26. OIE, 2017b. Immediate notifications and follow-ups. Tilapia lake virus, the Philippines. Retrieved from https://www.oie.int/wahis_2/public/wahid.php/Revie wrepo rt/Revie w?page_refer =MapFu llEve ntReport&repor tid=25278.

27. OIE, 2018. Immediate notification. Tilapia lake virus. Mexico. Retrieved from https://www.oie.int/wahis_2/publi c/wahid.php/Revie wreport/Revie w? page_refer =MapFu llEve ntRep ort&repor tid=27650.

28. Paria, A., Yadav, S.C., Verma, D.K., Mishra, A., Rastogi, A., Ravindra, Swaminathan, T.R., Rajendran, K.V., Sood, N. and Pradhan, P.K. 2023. Susceptibility of selected tropical non-tilapine ornamental cichlids to Tilapia tilapinevirus following experimental infection. Aquaculture, 567:739224. https://doi.org/10.1016/j.aquaculture.2022.739224. DOI: https://doi.org/10.1016/j.aquaculture.2022.739224

29. Phusantisampan, T., Tattiyapong, P., Mutrakulcharoen, P., Sriariyanun, M. and Surachetpong, W. 2019. Rapid detection of tilapia lake virus using a one-step reverse transcription loop-mediated isothermal amplification assay. Aquaculture 507:35-39. https://doi.org/10.1016/j.aquaculture.2019.04.015. DOI: https://doi.org/10.1016/j.aquaculture.2019.04.015

30. Pierezan, F., Yun, S., Piewbang, C., Surachetpong, W. and Soto, E. 2020. Pathogenesis and immune response of Nile tilapia (Oreochromis niloticus) exposed to Tilapia lake virus by intragastric route. Fish. Shellfish Immunol., 107:289-300. https://doi.org/10.1016/j.fsi.2020.10.019. DOI: https://doi.org/10.1016/j.fsi.2020.10.019

31. Pierezan, F., Yun, S., Surachetpong, W. and Soto, E. 2019. Intragastric and intracoelomic injection challenge models of tilapia lake virus infection in Nile tilapia (Oreochromis niloticus L.) fingerlings. J. Fish Dis., 42(9):1301-1307. https://doi.org/10.1111/jfd.13052. DOI: https://doi.org/10.1111/jfd.13052

32. Pulido, L.L.H., Mora, C.M., Hung, A.L., Dong, H.T. and Senapin, S. 2019. Tilapia lake virus (TiLV) from Peru is genetically close to the Israeli isolates. Aquaculture, 510:61-65. https://doi.org/10.1016/j.aquaculture.2019.04.058. DOI: https://doi.org/10.1016/j.aquaculture.2019.04.058

33. Rakus, K., Mojzesz, M., Widziolek, M., Pooranachandran, N., Teitge, F., Surachetpong, W., Chadzinska, M., Steinhagen, D. and Adamek, M. 2020. Antiviral response of adult zebrafish (Danio rerio) during tilapia lake virus (TiLV) infection. Fish. Shellfish Immunol., 101:1-8. https://doi.org/10.1016/j.fsi.2020.03.040 DOI: https://doi.org/10.1016/j.fsi.2020.03.040

34. Rao, M., Kumar, S.H., Kumar, S., Bedekar, M.K., Tripathi, G. and KoolothValappil, R. 2021. Microbiological investigation of Tilapia lake virus-associated mortalities in cage-farmed Oreochromis niloticus in India. Aquacult. Int., 29(2):511-526. https://doi.org/10.1007/s10499-020-00635-9. DOI: https://doi.org/10.1007/s10499-020-00635-9

35. Senapin, S., Shyam, K.U., Meemetta, W., Rattanarojpong, T. and Dong, H.T. 2018. Inapparent infection cases of tilapia lake virus (TiLV) in farmed tilapia. Aquaculture, 487:51-55. https://doi.org/10.1016/j.aquaculture.2018.01.007. DOI: https://doi.org/10.1016/j.aquaculture.2018.01.007

36. Surachetpong, W., Janetanakit, T., Nonthabenjawan, N., Tattiyapong, P., Sirikanchana, K. and Amonsin, A. 2017. Outbreaks of tilapia lake virus infection, Thailand, 2015-2016. Emerg. Infect. Dis., 23(6):1031. https://doi.org/10.3201%2Feid2306.161278. DOI: https://doi.org/10.3201/eid2306.161278

37. Taengphu, S., Sangsuriya, P., Phiwsaiya, K., Debnath, P.P., Delamare-Deboutteville, J., Mohan, C.V., Dong, H.T. and Senapin, S. 2020. Genetic diversity of tilapia lake virus genome segment 1 from 2011 to 2019 and a newly validated semi-nested RT-PCR method. Aquaculture, 526:735423. https://doi.org/10.1016/j.aquaculture.2020.735423. DOI: https://doi.org/10.1016/j.aquaculture.2020.735423

38. Tajadini, M., Panjehpour, M. and Javanmard, S.H. 2014. Comparison of SYBR Green and TaqMan methods in quantitative real-time polymerase chain reaction analysis of four adenosine receptor subtypes. Adv. Biomed. Res., 3:85. https://doi.org/10.4103/2277-9175.127998. DOI: https://doi.org/10.4103/2277-9175.127998

39. Tattiyapong, P., Sirikanchana, K. and Surachetpong, W. 2018. Development and validation of a reverse transcription-quantitative polymerase chain reaction for tilapia lake virus detection in clinical samples and experimentally challenged fish. J. Fish Dis., 41(2):255-261. https://doi.org/10.1111/jfd.12708. DOI: https://doi.org/10.1111/jfd.12708

40. Tran, T.H., Nguyen, V.T.H., Bui, H.C.N., Tran, Y.B.T., Tran, H.T.T., Le, T.T.T., Vu, H.T.T. and Ngo, T.P.H. 2022. Tilapia Lake Virus (TiLV) from Vietnam is genetically distantly related to TiLV strains from other countries. J. Fish Dis., 45(9):1389-1401. https://doi.org/10.1111/jfd.13669. DOI: https://doi.org/10.1111/jfd.13669

41. Tsofack, J.E.K., Zamostiano, R., Watted, S., Berkowitz, A., Rosenbluth, E., Mishra, N., Briese, T., Lipkin, W.I., Kabuusu, R.M., Ferguson, H. and Del Pozo, J. 2017. Detection of tilapia lake virus in clinical samples by culturing and nested reverse transcription-PCR. J. Clin. Microbiol., 55(3):759-767. https://doi.org/10.1128/jcm.01808-16. DOI: https://doi.org/10.1128/JCM.01808-16

42. Waiyamitra, P., Tattiyapong, P., Sirikanchana, K., Mongkolsuk, S., Nicholson, P. and Surachetpong, W. 2018. A TaqMan RT-qPCR assay for tilapia lake virus (TiLV) detection in tilapia. Aquaculture, 497:184-188. https://doi.org/10.1016/j.aquaculture.2018.07.060. DOI: https://doi.org/10.1016/j.aquaculture.2018.07.060

43. Wang, Y., Wang, Q., Bergmann, S.M., Li, Y., Li, B., Lv, Y., Yin, J., Yang, G., Qv, Y., Wang, Y. and Zeng, W. 2021. Development and comparative evaluation of real-time PCR and real-time RPA assays for detection of tilapia lake virus. Mol. Cell. Probes, 60:101776. https://doi.org/10.1016/j.mcp.2021.101776. DOI: https://doi.org/10.1016/j.mcp.2021.101776

44. WOAH, 2022. Infection with tilapia lake virus- A novel Orthomyxo-like virus. https://www.woah.org/app/uploads/2022/11/a-woah-tilv-disease-card-sept-2022.pdf.

45. Yadav, M.K., Rastogi, A., Criollo Jaoquin, M.P., Verma, D.K., Rathore, G., Swaminathan, T.R., Paria, A., Pradhan, P.K. and Sood, N. 2021. Establishment and characterization of a continuous cell line from heart of Nile tilapia Oreochromis niloticus and its susceptibility to tilapia lake virus. J. Virol. Methods, 287:113989. https://doi.org/10.1016/j.jviromet.2020.113989. DOI: https://doi.org/10.1016/j.jviromet.2020.113989

46. Yadav, R., Paria, A., Mankame, S., Makesh, M., Chaudhari, A. and Rajendran, K.V. 2015. Development of SYBR Green and TaqMan quantitative real-time PCR assays for hepatopancreatic parvovirus (HPV) infecting Penaeus monodon in India. Mol. Cell. Probes, 29:442-448. https://doi.org/10.1016/j.mcp.2015.07.006. DOI: https://doi.org/10.1016/j.mcp.2015.07.006

47. Yamkasem, J., Tattiyapong, P., Kamlangdee, A. and Surachetpong, W. 2019. Evidence of potential vertical transmission of tilapia lake virus. J. Fish Dis., 42(9):1293-1300. https://doi.org/10.1111/jfd.13050. DOI: https://doi.org/10.1111/jfd.13050

48. Yin, J., Wang, Q., Wang, Y., Li, Y., Zeng, W., Wu, J., Ren, Y., Tang, Y., Gao, C., Hu, H. and Bergmann, S.M. 2019. Development of a simple and rapid reverse transcription-loop-mediated isothermal amplification (RT‐LAMP) assay for sensitive detection of tilapia lake virus. J. Fish Dis., 42(6):817-824. https://doi.org/10.1111/jfd.12983. DOI: https://doi.org/10.1111/jfd.12983