Abstract
Tilapia lake virus (TiLV) is a novel single-stranded RNA virus that is considered a threat to the universal tilapia industry. Recombinant technology proved to apply to different viruses and does not require handling live viruses. The present study developed a recombinant protein from segment 4 of TiLV with the aim of developing an immunological detection method for the virus. For this purpose, the complete coding sequence of segment 4 of the virus was optimized, amplified, and cloned in pET-28a( +), a prokaryotic expression vector, and the TiLV segment 4 construct (pET-seg4) was developed. The recombinant protein was expressed in BL21-competent Escherichia coli by isopropyl-β-D-thiogalactopyranoside (IPTG) induction. The successful expression of the recombinant protein was confirmed by SDS-PAGE. The polyclonal antibody (PAb) raised against the recombinant protein was used for the indirect enzyme-linked immunosorbent assay (ELISA) development to detect TiLV in the pooled kidney and mucus samples. The coating concentrations for the recombinant protein and the TiLV-positive samples were standardized as 3.125 µg and 6.25 µg, respectively, and the optimum polyclonal antibody dilution was found as 1:800. The diagnostic sensitivity and diagnostic specificity of the assay were 82.35% and 100%, respectively. The recombinant protein developed in this study provided a better understanding of raising a codon-optimized protein and its use in developing an indirect ELISA test for TiLV, which can also be used as a recombinant vaccine for immunizing tilapia.
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References
Abu Rass R, Kustin T, Zamostiano R, Smorodinsky N, Ben Meir D, Feder D, Mishra N, Lipkin WI, Eldar A, Ehrlich M, Stern A (2022) Inferring protein function in an emerging virus: detection of the nucleoprotein in tilapia lake virus. J Virol 96(6):e01757-e1821
Acharya V, Chakraborty HJ, Rout AK, Balabantaray S, Behera BK, Das BK (2019) Structural characterization of open reading frame-encoded functional genes from tilapia lake virus (TiLV). Mol Biotechnol 61:945–957. https://doi.org/10.3390/jmse10010100
Bacharach E, Mishra N, Briese T, Zody MC, Tsofack JEK, Zamostiano R, Berkowitz A, Ng J, Nitido A, Corvelo A, Toussaint NC (2016) Characterization of a novel orthomyxo-like virus causing mass die-offs of tilapia. Mbio 7(2):e00431-e516. https://doi.org/10.1128/mBio.00431-16
Bakopoulos V, Volpatti D, Papapanagiotou E, Richards R, Galleotti AA (1997) Development of an ELISA to detect Pasteurella piscicida in culture and in ‘spiked’ fish tissue. Aquaculture 156:359–366
Bedekar MK, Kole S, Tripathi G (2020) Biotechnological approaches to fish vaccine. In: Genomics and biotechnological advances in veterinary, poultry, and fisheries. Academic Press, pp 407–419. https://doi.org/10.1016/B978-0-12-816352-8.00017-5
Behera BK, Pradhan PK, Swaminathan TR, Sood N, Paria P, Das A, Verma DK, Kumar R, Yadav MK, Dev AK, Parida PK (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
Bernhofer M, Dallago C, Karl T, Satagopam V, Heinzinger M, Littmann M, Olenyi T, Qiu J, Schütze K, Yachdav G, Ashkenazy H (2021) PredictProtein-predicting protein structure and function for 29 years. Nucleic Acids Res 49:W535–W540
Classen DC, Morningstar JM, Shanley JD (1987) Detection of antibody to murine cytomegalovirus by enzyme-linked immunosorbent and indirect immunofluorescence assays. Journal of Clinical Microbiology 25:600–4. https://doi.org/10.1128/jcm.25.4.600-604.1987
Criollo-Joaquin M, Motte E, Salvatierra M, Medina J, Diringer B, Sandoval G, Mialhe E (2019) Design and evaluation of the expression of a potential vaccine of DNA against lake tilapia virus (TiLV). Rev Peru Biol 26:301–310
Dixon PF, Hill BJ (1984) Rapid detection of fish rhabdoviruses by the enzyme-linked immunosorbent assay (ELISA). Aquaculture 42:1–12
Dong HT, Ataguba GA, Khunrae P, Rattanarojpong T, Senapin S (2017a) Evidence of TiLV infection in tilapia hatcheries from 2012 to 2017 reveals probable global spread of the disease. Aquaculture 479:579–583. https://doi.org/10.1016/j.aquaculture.2017.06.035
Dong HT, Siriroob S, Meemetta W, Santimanawong W, Gangnonngiw W, Pirarat N, Khunrae P, Rattanarojpong T, Vanichviriyakit R, Senapin S (2017b) 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
Eichelberger MC, Morens DM, Taubenberger JK (2018) Neuraminidase as an influenza vaccine antigen: a low hanging fruit, ready for picking to improve vaccine effectiveness. Curr Opin Immunol 53:38–44. https://doi.org/10.1016/j.coi.2018.03.025
Eyngor M, Zamostiano R, Tsofack JEK, 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
FAO (2018) The State of World Fisheries and Aquaculture 2018 (SOFIA). Meeting the sustainable development goals Rome. Available from: https://www.fao.org/documents/card/en?details=I9540EN. Accessed 14 Aug 2023
Fathi M, Dickson C, Dickson M, Leschen W, Baily J, Muir F, Ulrich K, 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
Ferguson HW, Kabuusu R, Beltran S, Reyes E, Lince JA, 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
Ghaffari AD, Dalimi A, Ghaffarifar F, Pirestani M (2020) Structural predication and antigenic analysis of ROP16 protein utilising immunoinformatics methods in order to identification of a vaccine against Toxoplasma gondii: an in silico approach. Microb Pathog 142:104079. https://doi.org/10.1016/j.micpath.2020.104079
Goldman E, Rosenberg AH, Zubay G, Studier WF (1995) Consecutive low-usage leucine codons block translation only when near the 5′ end of a message in Escherichia coli. J Mol Biol 245:467–473. https://doi.org/10.1006/jmbi.1994.0038
Gupta P, Waheed SM, Bhatnagar R (1999) Expression and purification of the recombinant protective antigen of Bacillus anthracis. Protein Expr Purif 16:369–376
Hasan A, Hossain M, Alam J (2013) A computational assay to design an epitope-based peptide vaccine against Saint Louis encephalitis virus. Bioinform Biol Insights 7:BBI-S13402. https://doi.org/10.4137/BBI.S13402
Hopp TP, Woods KR (1981) Prediction of protein antigenic determinants from amino acid sequences. Proc Natl Acad Sci 78:3824–3828. https://doi.org/10.1073/pnas.78.6.3824
Hu H, Zeng W, Wang Y, Wang Q, Bergmann SM, Yin J, Li Y, Chen X, Gao C, Zhang D, Liu C (2020) Development and application of a recombinant protein-based indirect ELISA for detection of anti-tilapia lake virus IgM in sera from tilapia. Aquaculture 520:734756. https://doi.org/10.1016/j.aquaculture.2019.734756
International Committee on the Taxonomy of Viruses (ICTV) (2018) Virus taxonomy: 2018b Release. Available from: https://ictv.global/taxonomy/taxondetails?taxnode_id=202206025&taxon_name=Amnoonviridae
Jansen MD, Dong HT, Mohan CV (2018) Tilapia lake virus: a threat to the global tilapia industry? Rev Aquac. https://doi.org/10.1111/raq.12254
Kane JF (1995) Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli. Curr Opin Biotechnol 6:494–500. https://doi.org/10.1016/0958-1669(95)80082-4
Kathwate GH (2022) In silico design and characterisation of multi-epitopes vaccine for SARS-CoV2 from its spike protein. Int J Pept Res Ther 28:1–15. https://doi.org/10.1007/s10989-021-10348-z
KembouTsofack JE, Zamostiano R, Watted S, Berkowitz A, Rosenbluth E, Mishra N, Briese T, Lipkin WI, Kabuusu RM, Ferguson H, Del Pozo J (2017) Detection of tilapia lake virus in clinical samples by culturing and nested reverse transcription-PCR. J Clin Microbiol 55:759–767. https://doi.org/10.1128/JCM.01808-16
Kohl TO, Ascoli CA (2017) Indirect immunometric ELISA. Cold Spring Harb Protoc 2017:396–402. https://doi.org/10.1101/pdb.prot093708
Kolaskar AS, Tongaonkar PC (1990) A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS Lett 276:172–174. https://doi.org/10.1016/0014-5793(90)80535-Q
Koren CWR, Nylund A (1997) Morphology and morphogenesis of infectious salmon anaemia virus replicating in the endothelium of Atlantic salmon Salmo salar. Dis Aquat Org 29:99–109. https://doi.org/10.3354/dao029099
Kumari R, Kole S, Soman P, Rathore G, Tripathi G, Makesh M, Rajendran KV, Bedekar MK (2018) Bicistronic DNA vaccine against Edwardsiella tarda infection in Labeo rohita: construction and comparative evaluation of its protective efficacy against monocistronic DNA vaccine. Aquaculture 485:201–209. https://doi.org/10.1016/j.aquaculture.2017.11.052
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685
Lin AV (2015). Indirect Elisa. ELISA: methods and protocols, 51–59. https://doi.org/10.1007/978-1-4939-2742-5_5
Lueangyangyuen A, Senapin S, Dong HT, Unajak S, Wangkahart E, Khunrae P (2022) Expression and purification of S5196–272 and S6200–317 proteins from tilapia lake virus (TiLV) and their potential use as vaccines. Protein Expr Purif 190:106013
Leya T, Ahmad I, Valappil RK, Kurcheti PP, Tripathi G, Sharma R, Bedekar MK (2021) Development of species-specific IgM antibodies and elevation of mucosal immune response in Labeo rohita using recombinant bicistronic nano DNA vaccine priming. Fish & Shellfish Immunol 113:185–95. https://doi.org/10.1016/j.fsi.2021.04.008
Mauro VP (2018) Codon optimization in the production of recombinant biotherapeutics: potential risks and considerations. BioDrugs 32:69–81. https://doi.org/10.1007/s40259-018-0261-x
Mikalsen AB, Sindre H, Torgersen J, Rimstad E (2005) Protective effects of a DNA vaccine expressing the infectious salmon anemia virus hemagglutinin- esterase in Atlantic salmon. Vaccine 23(41):4895–4905. https://doi.org/10.1016/j.vaccine.2005.05.025
Nicholson P, Fathi MA, Fischer A, Mohan C, Schieck E, Mishra N, Heinimann A, Frey J, Wieland B, Jores J (2017) Detection of tilapia lake virus in Egyptian fish farms experiencing high mortalities in 2015. J Fish Dis 40(12):1925–1928. https://doi.org/10.1111/jfd.12650
Olesen NJ, Jørgensen PEV (1991) Rapid detection of viral haemorrhagic septicaemia virus in fish by ELISA. J Appl Ichthyol 7(3):183–186
Papaleo E, Saladino G, Lambrughi M, Lindorff-Larsen K, Gervasio FL, Nussinov R (2016) The role of protein loops and linkers in conformational dynamics and allostery. Chem Rev 116:6391–6423
Pulido LLH, Mora CM, Hung AL, Dong HT, 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
Rajendran KV, Sood N, Rao BM, Valsalam A, Bedekar MK, Jeena K, Pradhan PK, Paria A, Swaminathan TR, Verma DK, Sood NK (2023) Widespread occurrence of tilapia parvovirus in farmed Nile tilapia Oreochromis niloticus from India. J Fish Dis. https://doi.org/10.1111/jfd.13871
Rodiansyah A, Tan MI, Nugrahapraja H (2022) Construction, cloning, and overexpression of Staphylococcal Enterotoxin B Gene Synthetic (SEBsyn) in pET-28a (+): Pre-development Bacterial-Toxin Therapy for Cancer. In 7th International Conference on Biological Science (ICBS 2021) (pp. 464–470). Series: Advances in Biological Sciences Research, Atlantis Press. ISSN: 2468–5747. https://doi.org/10.2991/absr.k.220406.065
Sahdev S, Khattar SK, Saini KS (2008) Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies. Mol Cell Biochem 307:249–264. https://doi.org/10.1007/s11010-007-9603-6
Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J, Thompson JD (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:539. https://doi.org/10.1038/msb.2011.75
Subramaniam K, Ferguson HW, Kabuusu R, Waltzek TB (2019) Genome sequence of tilapia lake virus associated with syncytial hepatitis of tilapia in an Ecuadorian aquaculture facility. Microbiol Resour Announc 8:e00084-e119
Surachetpong W, Janetanakit T, Nonthabenjawan N, Tattiyapong P, Sirikanchana K, Amonsin A (2017) Outbreaks of tilapia lake virus infection, Thailand, 2015–2016. Emerg Infect Dis 23:1031. https://doi.org/10.3201/eid2306.161278
Tattiyapong P, Dechavichitlead W, Waltzek TB, Surachetpong W (2020) Tilapia develop protective immunity including a humoral response following exposure to tilapia lake virus. Fish Shellfish Immunol 106:666–674
United Nations Department of Economic and Social Affairs Population Division (2019) World population prospects 2019: Highlights. United Nations. Retrieved from from: https://population.un.org/wpp/Publications/Files/WPP2019_Highlights.pdf
Valsalam A, Rajendran KV, Kezhedath J, Godavarikar A, Sood N, Bedekar MK (2023) Development of an indirect ELISA test for the detection of tilapia lake virus (TiLV) in fish tissue and mucus samples. Microb Pathog 315:114707
Valsalam A, Bedekar MK, Kezhedath J, Sood N, Poojary N, Namdeo MS, Shrivastava N, Rajendran KV (2024) Isolation, in vitro, and in vivo pathogenicity test of tilapia lake virus (TiLV) and development of a prognostic semi-quantitative lesion scoring system for differentiating clinical/subclinical infection in farmed tilapia (Oreochromis niloticus L.). Microb Pathog 186:106475
Yadava A, Ockenhouse CF (2003) Effect of codon optimization on expression levels of a functionally folded malaria vaccine candidate in prokaryotic and eukaryotic expression systems. Infect Immun 71:4961–4969. https://doi.org/10.1128/IAI.71.9.4961-4969.2003
Zeng W, Wang Y, Chen X, Wang Q, Bergmann SM, Yang Y, Wang Y, Li BO, Lv Y, Li H, Lan W (2021) Potency and efficacy of VP20-based vaccine against tilapia lake virus using different prime-boost vaccination regimens in tilapia. Aquaculture 539:736654
Acknowledgements
The authors acknowledge the Director, ICAR-Central Institute of Fisheries Education, Mumbai, India, for providing all the essential facilities and the DBT, GOI.
Funding
The funding was provided by ICAR-Central Institute of Fisheries Education and Department of Biotechnology (BT/PR30609/AAQ/03/939/2018), GOI, India.
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LF- bench works and writing, data compilation. MKB- Conceptualization, data analysis and interpretation. AG- bench works, data compilation and writing. AV- Methodology, data compilation; Writing- original draft preparation. GBP- Reviewing and Editing. RKV-Monitoring; Writing-Correction.
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Lalruatfela, Bedekar, M.K., Godavarikar, A. et al. Molecular cloning and expression of codon-optimized segment 4 hypothetical protein (35 kDa) of tilapia lake virus (TiLV) in pET-28a( +) expression vector and development of indirect ELISA test. Aquacult Int (2024). https://doi.org/10.1007/s10499-024-01452-0
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DOI: https://doi.org/10.1007/s10499-024-01452-0