Development and validation of a synthetic peptide-based field deployable latex slide agglutination test for detection of Tilapia tilapinevirus

Meshram Supradhnya Namdeo; Megha Kadam Bedekar; Jeena Kezhedath; Kooloth Valappil Rajendran; Anisha Valsalam; Ankita Godavarikar; Pooja Vinde; Sagar Hundare; Khangembam Victoria Chanu; Dimpal Thakuria; Narrotam P. Sahu

doi:10.1371/journal.pone.0344743

Abstract

Tilapia significantly contributes to global food security and is an affordable protein source for most developing nations. Tilapia tilapinevirus (TiLV) poses a significant economic threat to the global tilapia industry. This study aimed to develop a rapid and accurate detection method for TiLV by synthesizing a monoclonal antibody (MAb) against it. A novel peptide, KLH-CQ, derived from the TiLV sequence, was designed considering physicochemical properties like net cationic charge, amphipathicity, helicity, and hydrophobicity. The KLH-CQ (50 µg) was used to immunize Balb/c mice with Freund's complete adjuvant. The presence of specific antibodies in the mice serum was confirmed by ELISA, which showed a high antibody titre of 2.67:0.12 (mean OD of treated Vs control sera). The mouse with the strongest immune response was used for spleen donor in hybridoma production. Epitope mapping via ELISA screening identified five positive clones (TiLV-MAb 1–5), with the most reactive clone selected for further analysis. Using Classen's method, a cutoff OD value of 1.24 ± 0.45 was determined for virus detection. The selected TiLV-MAb was then used as a probing antibody to develop a latex slide agglutination assay (TiLV-LAT) using passive adsorption method. Validation of the assay with tissue and mucus samples revealed a specificity of 88.37% and a sensitivity of 82.37% for TiLV detection. The overall accuracy of the assay was 83.51%, with positive and negative likelihood ratios of 7.06 and 0.2, respectively. The TiLV-LAT successfully detected TiLV in various tissues, showing variable sensitivity: liver (77.35%), mucus (73.53%), brain (67.92%), and kidney (62.26%). TiLV-LAT developed here has minimized the tedious steps involved in nucleic acid-based detection assays, with the recorded sensitivity and specificity; it can be used as a presumptive diagnosis for testing and point of care/farm site. Moreover, non-lethal sampling and virus testing in mucus samples would be useful for fish health monitoring.

Citation: Supradhnya Namdeo M, Bedekar MK, Kezhedath J, Rajendran KV, Valsalam A, Godavarikar A, et al. (2026) Development and validation of a synthetic peptide-based field deployable latex slide agglutination test for detection of Tilapia tilapinevirus. PLoS One 21(3): e0344743. https://doi.org/10.1371/journal.pone.0344743

Editor: Bijay Kumar Behera, Ministry of Fisheries, Animal Husbandry and Dairying, INDIA

Received: August 25, 2025; Accepted: February 24, 2026; Published: March 12, 2026

Copyright: © 2026 Supradhnya Namdeo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The data supporting the findings of this study are fully included in the manuscript.

Funding: The study was a part of the project funded by Department of Biotechnology (DBT), Government of India (BT/PR30609/AAQ/03/939/2018).The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have affirmed the absence of any competing interests.

Introduction

Tilapia significantly contributes to global food security and is an affordable protein source for most developing nations. Often referred to as “aquatic chicken” due to its low priced compared with other food fish [1]. In 2024, the worldwide production reached 7 million tonnes, an increase of 4–5 percent over 2023 [2]. Even though tilapia are recognized as a “hardy species” [3], in recent times, tilapia have been reportedly susceptible to several bacterial and viral diseases in aquaculture [4]. The regular encountered pathogens included Aeromonas veronii, A. hydrophila, A. caviae, A. jandaei, Edwardsiella tarda, Streptococcus agalactiae, S. iniae, Flavobacterium columnare, Lactococcus garviae, Pseudomonas spp., Ichthyophitirius multifiliis, Tricodina sp., Gyrodactylus niloticus, Tilapia tilapinevirus (TiLV) and Tilapia parvovirus (TiPV) [5–11]. Among the prevalent diseases that cause high mortality rates, TiLV infection is one of them, which impacts profitability and productivity. Since 2009, a drop in tilapia production was observed in Israel, but the cause was unexplained, later confirmed in 2014, i.e., Tilapinevirus tilapia [7]. The rate of morbidity and mortality stated around 0–100% with variable geographical location, such as 10–80% was noted in Ecuadorian tilapia nurturing systems [12], 6.4% at Chinese Taipei [13], and 80–90% in India [4]. From fry to broodstock, all life stages were susceptible to TiLV infection [7]. Due to this virus, 98,000 tons of yearly yield losses were estimated at $ 100 million in Egypt [14].

Numerous diagnostic methods have been developed for TiLV identification including cell culture, histopathology, in-situ hybridization [7], reverse transcriptase-polymerase chain reaction, (RT-PCR- nested and semi-nested) [15], reverse transcription LAMP [16,17], quantitative real-time PCR (qRT-PCR) [18], iELISA using the recombinant protein of segment eight of the virus [19], ELISA to detect serum IgM antibody against TiLV [20]. Some of the cell lines for virus isolation or susceptibility are E-11 from Ophicephalus striatus (cloned of SSN-1) [7], TmB and OmB from O. mossambicus [21], OnlB and OnlL from Oreochromis niloticus [22], and CFF from Pristolepis fasciatus [4].

Direct detection of TiLV viral particles (antigens) using polyclonal IgG from tissue by immunohistochemistry (IHC) [23] and ELISA has shown the potential of antigen-antibody testing of the virus in mucus and other tissues [24]. Here we aimed to develop an antigen-antibody based field deployable latex agglutination test for TiLV testing (TiLV-LAT), where lab developed monoclonal antibodies against segment 8 were coupled with latex beads and used as testing reagent.

Materials and methods

Animal ethics statement

The study conformed with current animal welfare regulations and was approved by the Board of Studies and authorities of the ICAR-Central Institute of Fisheries Education, Mumbai, India. All experimental procedures and the monoclonal antibody (MAb) production were carried out with the approval of the Institutional Biosafety Committee (IBSC), approval no F.No.CIFE/IBSC/2022/6 and Institute Animal Ethics Committee (IAEC) of CIFE approval no CIFE-03–2019.

Designing, synthesis and purification of peptide

TiLV, segment 8 gene (complete coding sequence), were retrieved from NCBI, and alignment was performed in ClustalW [25]. Indian origin sequence (accession no MZ297930.1) was selected for a novel peptide design. The most conserved segment of the sequence showing the highest immunogenic score (0.952) was selected from 5 designed peptides. An antigenicity prediction of the selected fragment was made by the IEDB analysis program (http://tools.iedb.org/ellipro/result/predict/). The novel designed peptide was synthesized by GL Biochem Ltd, Shanghai, China. Purification was done by reversed phase-high performance liquid chromatography (RP-HPLC) and molar mass was estimated through MALDI-TOF (Agilent-6125B).

An in silico evaluation of the novel peptide KLH-CQ was performed utilizing several online tools, including a peptide property calculator (https://pepcalc.com/) to assess hydrophobicity, charge, solubility, and HLP (http://crdd.osdd.net/raghava/hlp/pep_both.htm) for further peptide characteristics. The secondary structure was analyzed by PSI-PRED [26] and GOR4 [27]. Three-dimensional structure of the peptide was created using PEP-FOLD3 [28] and visualized by Pymol. Wenxiang web server [29] used to create wexiang diagram and NetWheels server [30] for helical wheel and net projections.

Immunization

The synthesized TiLV segment 8 gene (KLH-CQ) peptide was used as an antigen for immunization. Two Balb/c mice (8 weeks old) were purchased from Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Kharghar, Maharashtra and maintained in cages in a well-ventilated animal room, CIFE, Mumbai. Mice were intraperitoneally immunized with 50 µg TiLV (KLH-CQ) mixed with Freund's complete adjuvant (Sigma, USA) per mouse. The animals were boosted on the 14^th and 28^th day, with the same dose intraperitoneally emulsified with Freund's incomplete adjuvant (Sigma, USA). Ten days after the second booster dose, the mice were bled and blood collected. The separated serum was used in ELISA to determine the antibody titer. On the 36^th day, the mice with high antibody titer were injected with 50 µg peptide intravenously. Three days after the intravenous booster, the mouse was bled and the sera were separated, aliquoted, labelled and preserved at −20°C for further analysis.

Monoclonal antibody generation

The cell fusion method was adopted from the method developed by [31] with some modification. In short, myeloma cells SP2/0 (ATCC) were grown in Iscove's Modiﬁed Dulbecco's Medium (IMDM) containing 15% fetal bovine serum (FBS) at 37°C with 5% CO_2. After forming the monolayer, the cells were sub-cultured to maintain the logarithmic growth phase. The spleen cells from the immunized mouse were fused with SP2/0 myeloma cell line using (50%) polyethylene glycol (PEG; Gibco, USA). The fused cells were resuspended in IMDM with 20% FBS supplemented with 2X HAT (Hypoxanthine Aminopterin Thymidine) and seeded into the 96-well tissue culture plate containing feeder cells (1 × 10⁵ cells/well) at the rate of 100 µl per well and incubated in a 5% CO₂ incubator. After two weeks, supernatants from the wells were screened for antibodies by indirect ELISA. The agglutination assay mentioned below screened the positive clones for virus specificity and cross-reactivity. The desired clones were cloned by the limiting dilution method, modified from [32] and cryopreserved.

Sample collection

Nile tilapia (Oreochromis niloticus L.) was collected from various geographic locations of Maharashtra state, India, where a disease outbreak occurred at different time points (February 2022 to April 2023). The tilapia fish (n = 1200) were approximately 11 ± 0.5 cm and 10 ± 2 g, apparently healthy fish were brought and maintained with biosecurity in the wet lab of the Aquatic Animal Health Laboratory, ICAR- Central Institute of Fisheries Education (CIFE), Mumbai, Maharashtra, India. Both diseased and apparently healthy fish were anesthetized with MS-222 (Sigma-Aldrich, USA) (50 mg L^-1) by the immersion method, following cessation of opercular movement. The sampling was done ethically following the CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals, Government of India) regulation. The clinical and post-mortem observation were noted and sampled separately by dissection of the organs (liver, kidney, brain) and mucus (fish washed with phosphate saline buffer (PBS) in sterile plastic bag and collected) were pooled (n = 5 each organ/mucus) and preserved in the Trizol reagent (Invitrogen) for subsequent analysis meanwhile the same organs was collected in Lebovitz- L (L-15) (Gibco, USA) medium with 2% fetal bovine serum (FBS) and 1% antibiotic and antimycotic (Gibco, USA) and kept in −80°C for virus isolation. The remaining fish were immediately transferred to separate, clean, and highly aerated water and monitored until they regained normal behaviour.

RNA isolation and cDNA synthesis

The viral RNA was extracted from pooled organs using QIAamp viral RNA kit, Qiagen as per the developer's protocol. The viral RNA was quantified (Nanodrop 2000 spectrophotometer, Thermo Fisher Scientific, USA) and purity checked before converted to complementary DNA (cDNA). The cDNA was synthesized using random primers obtained with Revert Aid reverse transcriptase First Strand cDNA Synthesis kit (Thermo Fisher, USA) and stored at −20°C.

RT-PCR detection

The TiLV detection was performed by semi-nested RT-PCR of segment 3 [15,24], and segment 8 [33]. Each PCR reaction volume of 20 μl contained 2X master mix (Emerald Amp, Takara) 10μl, 0.4μl (0.2μM) of forward and reverse primer and 1μl cDNA template, and the rest was nuclease-free water. TiLV positive sample (TiLV-MH-2022) was available in the Department of Aquatic Animal Health Management (AAHM), ICAR-CIFE, Mumbai [34] was used as positive controlled fallowed by PCR condition for segment 3 was 94°C for 2 min initial denaturation, 30 cycles of 94°C, 60°C and 72°C for 30 sec and final extension for 5 min at 72°C. Thermocycling conditions were adopted from [33] for segment 8 with modification as initial denaturation 94°C for 2 min, 35 cycles at 94°C for 30 sec, 55°C for 30 sec, and 68°C for 30 sec, followed by a final extension at 68°C for 5 min. The primer details were mentioned in Table 1.

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Table 1. Primers used in the current study.

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Virus isolation and identification

The OnlL and SSN-1 cells were used for virus isolation. The OnlL cell line was available in the department AAHM, ICAR-CIFE, Mumbai [34]; SSN-1 cell line was obtained from the Central Institute of Brackishwater Aquaculture (CIBA, Chennai). Both the cell lines were maintained with 10% FBS L-15 media with 1% antibiotic and antimycotic and passaged for each 4−5 days to reach 80−90% confluence. The stored samples (liver, kidney and brain) positive and negative were thawed on ice, homogenized in 1 ml 10% FBS L-15 media with 1% antibiotic and antimycotic solution. Homogenized tissues were centrifuged (6,700 × g for 10 min), filtered (0.22 μm PES filter), and the supernatant (250 μl) was used to infect 4-day-old confluent monolayers in a 25 cm² of OnlL and SSN-1 cells and incubated at 28°C and 25°C for 14 days. In the controlled flask, healthy tilapia (negative sample) filtrate was used and kept under the same parameters. The flasks were monitored daily for any cytopathic (CPE) changes. The virus susceptibility assay protocol was tailored from [34] and [35] for OnlL and SSN-1 cells. The virus was harvested, viral RNA extracted and confirmed through RT-PCR for its presence or absence for the generation of virus stock. Based on this, multiple flasks were infected, and the supernatant was recovered after post-infection and pooled. The virus precipitation protocol was adopted from [36]. In short, using 7% polyethylene glycol (PEG) 6000 and 2.3% NaCl, the virus was precipitated overnight at 4°C. Then it was pelleted by centrifugation at 13, 000 × g for 1 h. In order to remove the PEG, the pellet was centrifuged at 13,000 × g for 15 min after being resuspended in 1 ml of TES buffer (10 mM Tris-Cl [pH 7.4], 2 mM EDTA, 150 mM NaCl). The viral RNA was extracted from the supernatant, cDNA was synthesized and amplified. Amplicons were purified using a PCR purification kit (Thermo Scientific, USA), cloned with ClonJET PCR cloning kit (Thermo Scientific, USA) and sequenced using the Sanger direct sequencing method, a service provided by Eurofins Genomics, India.

Partial forward and reverse sequences of the segment 8 gene were assembled using BioEdit (version 7.2.5.0). A Basic Local Alignment Search Tool (BLAST) search was subsequently performed on NCBI to determine sequence identity against the entire database. Closely related sequences of TiLV segment 8 from various geographic locations were retrieved and subjected to multiple sequence alignment using ClustalW [25]. A phylogenetic tree was inferred using the neighbor-joining algorithm [37], implemented in Molecular Evolutionary Genetics Analysis (MEGA version 11) [25]. Evolutionary distances were calculated using the Kimura 2-parameter method [38], and bootstrap analysis with 1000 replicates was performed to assess the reliability of the tree groupings [39]. In total, 19 nucleotide sequences, including those from India, Thailand, Bangladesh, and Israel, were included in the phylogenetic analysis. The novel TiLV segment 8 strain SM-MH-2023 sequence was submitted to GenBank to obtain an accession number.

In vitro pathogenicity test and transmission electron microscopy (TEM)

The In vitro pathogenicity was performed in the two cell lines, OnlL and SSN-1 cells. A 96-well plate (Nunc™, Denmark) was seeded (1.5 × 10⁴ cells/well) with 200 μl of L-15 medium (5% FBS) per well to obtain 80% conﬂuence. Manual viable cell counting was performed with a hemocytometer using the following formula [40]: Viable cells mL^-1 = (Number of viable cell counted/Number of squares counted) × Dilution factor × 10⁴. The viral inoculum was serially diluted up to 100 in 5% L-15 medium. Then, 100 μl of each dilution was added to the wells and examined for 12 days. The TCID₅₀ mL^-1 was calculated using the Reed and Muench [41] method. Virus-infected OnlL cells were harvested, pelleted and washed with PBS (0.1 M phosphate-buffered saline), then fixed in 3% glutaraldehyde in 0.2 mol L^-1 sodium cacodylate buffer (pH 7.4). The post-processing was done at Jaslok Hospital and Research Centre, Mumbai, India, using an electron microscope at 80 kV (JEOL JEM 1010).

Preparation of TiLV-MAbs coated latex particles

White suspension of polystyrene latex beads, 0.8 mm in diameter, was purchased from Sigma Aldrich (LB8). TiLV-MAbs-latex reagents were prepared by the passive adsorption method adopted from [42], with some modifications. Briefly, a 100 µl latex beads suspension was mixed with 100 µl Glycine buffered saline, pH 8.2 (GBS: glycine 0.73% and 1% NaCl), containing 40 µl TiLV-MAbs. The mixture was incubated for 2 h at 37°C with periodic mixing. 200 µl Glysine buffered saline containing Bovine Serum Albumine (GBS-BSA: 0.1% final concentration of BSA to GBS) was added to the same mixture and blocked for 1 h at 37°C. The sensitized beads were stored for up to a year at 4°C.

Latex agglutination assay for TiLV detection (TiLV-LAT assay)

40 µL of PEG concentrated TiLV-positive tissue supernatant was placed on a glass slide, and 20 µl of TiLV-MAbs-latex beads were added as the testing reagent. PCR-negative tissue homogenate was employed as a negative control. The mixture was mixed gently and observed for an agglutination reaction for 3 min at room temperature. The relative degree of agglutination of TiLV was measured based on the area of clump formation with respect to the corresponding image. For semi-quantitative assessment, agglutination intensity was inspected and score was assigned from 1−4, in short 1 corresponds to small clumps, with 25% agglutination, 2 (50% agglutination), 3 (75% agglutination), and 4 (large clumps that forms in less than 2 min with 100% agglutination). For quantitative testing and sensitivity evaluation, the positive tissue homogenate sample was serially diluted into 2-fold (quantitative reference ranging from 1.35 × 10¹¹ to 4.22 × 10⁹ copies µl^-1). The cross reactivity was tested for other common pathogens of tilapia, such as nervous necrosis virus (NNV), tilapia parvovirus (TiPV) and some bacterial species, A. veronii, and S. agalactiae.

Performance evaluation of TiLV-LAT assay against semi-nested RT-PCR

The samples collected from different places in Maharashtra state, ranging from highly affected to healthy (apparently), were selected. Collectively, 194 pooled samples (149 tissue and 45 mucus) were tested by TiLV-LAT assay and semi-nested RT-PCR of segment 3 [15]. The correlation was studied between these two tests, and calculations were made, such as sensitivity and specificity.

Statistical analysis

The data were analyzed using the Microsoft Excel; GraphPad Prism8 and illustrations were drawn using Biorender software. TiLV⁺ samples with distinct agglutination scores and TiLV^- samples were analyzed using unpaired t-test with Welch’s correction (no assumption of equal SD between two groups).The values in the graphical representations were expressed as a mean ± standard deviation, and p values ≤ 0.05 were considered significant. The diagnostic test was estimated by MedCalc (https://www.medcalc.org/calc/diagnostic_test.php), and Cohen’s kappa is calculated (https://www.graphpad.com/quickcalcs/kappa1/).

Results

Synthesis and structure analysis of peptide

A synthetic peptide, KLH-CQ (CTNRKGQRFEFNRKQ-NH₂), consisting of 15 amino acids, was chosen based on an alignment analysis of segment 8 TiLV across 16 variants (Fig 1). KLH-CQ was synthesized using Fmoc-chemistry and purified via reverse-phase high-performance liquid chromatography (RP-HPLC) with a Boston Green 0DS-AQ column (250 × 4.6 mm). The detected molecular mass of the purified peptide was 1912.34 Da, which is close to the theoretical mass. The purity, determined by RP-HPLC, was > 85%. The mass spectrum and HPLC chromatogram of the synthesized KLH-CQ was generated. Secondary structure analysis confirmed the presence of both β structures and random coils, as determined by the PRED and GOR4 prediction methods (Fig 2) and other physicochemical parameters of KLH-CQ are given in Table 2. Additionally, the predicted 3D structure indicated the presence of helical and β sheets at both the N- and C-terminals (Fig 3a). This structure was further validated using the SAVES server, which confirmed that approximately 100% of the residues are in the favored region. Intramolecular bonding and the polarity of the residues were examined using wheel and net projections (Figs 3b and 3c). A Wenxiang diagram (Fig 3d) and helical wheel indicated that the KLH-CQ peptide is amphipathic, displaying both polar and non-polar residues. The net wheel illustrates specific interactions between residues adjacent to each other along the central axis of the helix in the peptide.

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Table 2. Physicochemical parameters of KLH-CQ peptide in inwards like environment.

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Fig 1. Multiple sequence alignment of the complete coding sequence of TiLV segment 8 protein showing conserved region (yellow colored) selected for designed peptide with modification.

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Fig 2. Secondary structure analysis of novel KLH-CQ peptide using PSI-PRED (A) and GOR4 (B) online tools.

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Fig 3. The complete coding sequence of TiLV segment 8 protein selected for designed peptide with modification; a: represents three-dimensional structure, b: the helical wheel structure, c: net projection, d: Wenxiang diagram of KLH-CQ peptide.

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Gross sign, PCR detection and isolation of TiLV

TiLV from infected tilapia showing clinical signs of skin erosion, haemorrhages all over the body and mouth, bilateral ocular protrusion (Fig 4) was confirmed through RT-PCR of segment 3 (semi-nested) and segment 8 prior to TiLV-LAT development of immune assay (Fig 5). Out of 212 pooled samples tested, the 167 samples were positive for segment 3, and 150 for segment 8. The prevalence of virus by test segment Vs tissue is illustrated in Fig 6. Notably, even in seemingly healthy fish, the semi-nested detection results demonstrated the presence of TiLV during the second round of amplification. The product from segment 8 PCR exhibited 99% nucleotide sequence identity with existing TiLV sequences (MZ297930.1) and has been submitted to GenBank under accession number PV470487. The diversity among the segment 8 were characterized by the evolutionary phylogenetic tree (Fig 7).

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Fig 4. Gross signs of infected tilapia, O. niloticus, showing distinct skin erosion, reddening of the mouth and body, discoloration of the body, bilateral ocular protrusion, ascites in body cavity, pale liver with blood congestion.

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Fig 5. Gel electrophoresis verification of the TiLV samples detected from apparently healthy and infected fish.

(A) Showing semi-nested RT-PCR of TiLV segment 3 in the apparently healthy fish (lanes 1-3 liver, kidney, brain) and infected fish (lanes 4-6 liver, 7-9 kidney and 10-12 brain) and mucus (13-15) samples. M: Molecular weight marker 100 bp plus. (B) Represents segment 8 pooled tissue sample (lanes 1-6) and mucus (lane 8 and 9). Each segment's desired basepair is mentioned on the positive control (PC) and the negative control (NC) (RNase-free water).

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Fig 6. The comparative performance of two RT-PCR based methods for detecting TiLV in field samples among the selected organs, one-way ANOVA determined significance difference (p < 0.05).

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Fig 7. The evolutionary relationship of segment 8 constructed by a neighbor-joining algorithm with a 1000 bootstrap value (scale 0.05), demonstrated close association among existing TiLV sequences.

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In the cell lines, OnlL and SSN-1, cytopathology was induced by TiLV field isolated virus, but early signs of cytopathic effect (CPE) were seen in SSN-1 cells, within 6 days post-infection compared to OnlL cells. The notable changes were cell elongation, plaque formation, cell detachment and absence of syncytium formation in SSN-1 cells (Fig 8 D-8F). In contrast, in OnlL cells, distinct syncytium and plaque formation were observed and the absence of cell detachment and limited cellular vacuole formation (Fig 8 A-8C). The virus supernatant and cells (pellet) were reconfirmed for the presence of the virus through segment 3 RT-PCR as a reference, suggested by Dong et al. [15], and tested positive (Fig 9). The median tissue infective dose (TCID₅₀) was calculated for both cell lines, and it was 1.73 × 10⁶ TCID₅₀ ml^-1 and 3.16 × 10⁷ TCID₅₀ ml^-1 for OnlL and SSN-1, respectively. Subsequently, TEM analysis revealed single, round, enveloped ribonucleoprotein complexes measuring 100–110 nm in diameter within the cytoplasm, consistent with TiLV (Fig 10).

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Fig 8. Isolation of virus from RT-PCR positive pooled tissue (liver, kidney, brain) homogenate was used in two cell lines.

(A) control OnlL cells; (B) infected cell line showing syncytial formation (black asterisk) with cell CPE and cell shrinkage (C) 12 days post-infection (dpi) at a magnification of 20X. SSN-1 cells inoculated with the same (D) control SSN-1 cells; (E & F) showing CPE with cell elongation (white arrow) and plaque formation (white asterisk), 6 dpi at a magnification of 20X.

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Fig 9. Represents a TiLV specific RT-PCR amplification in the infected OnlL (lanes 1-3) and SSN-1 (lanes 4-6), cells from culture, lane 3 and 6 is control of each cell line (1.8% agarose).

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Fig 10. Electron micrograph of ultrathin section of infected OnlL cells apparently showing ribonucleoprotein complexes (black arrow) with the envelope (~100 nm) in the first photograph, and electron dense dots in second picture show a typical appearance of virus particles within the cytoplasm.

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The virus was recovered from pooled samples (liver, kidney, brain) with clinical lesions routinely observed from these organs, this indicates the virus infection was systemic. The positive supernatant was utilized to develop a latex beads agglutination assay against monoclonal antibodies of TiLV (TiLV-MAbs).

Monoclonal antibody production and characterization

Epitope mapping was done by ELISA screening of the best clones generated from hybridoma against Tilapia tilapinevirus, and the peptide was taken as a positive control. Out of five positive clones of TiLV-MAb 1–5, which were selected by ELISA titre, the most reactive clone was selected for the screening of TiLV. The cutoff of the ELISA for virus detection was drawn by the Classen’s method, which was 1.24 ± 0.45 OD. The samples tested previously positive by PCR showed absolute specificity for TiLV-MAb. The clone detected TiLV in cell culture supernatant and tissue homogenate with absolute specificity.

TiLV detection by latex agglutination assay (TiLV-LAT assay)

A variety of viruses are detectable via latex beads agglutination assay utilizing specific antibodies. In principle, latex beads coated with specific antibodies would enable their respective agglutination (visible clumps) by the corresponding antigen (Fig 11). In ordered to examine this concept, we coated TiLV-MAbs to latex beads and incubated with TiLV positive sample, which led to strong agglutination within 3 min; conversely, the TiLV negative sample failed to induce agglutination, demonstrating that latex agglutination assay effectively distinguished TiLV positive from TiLV negative samples. Further, quantification was done both semi-quantitative and quantitative. In semi-quantitative agglutination, we assigned numerical scoring systems 1, 2, 3 and 4 for TiLV response. Based on the minimal background agglutination (0−4%) observed in TiLV-negative samples, we have set 5% agglutination as the cutoff for antibody positivity. The samples that produced 5% – 25% (1), 25% – 50% (2), 50% – 75% (3), and > 75% (4) agglutination, respectively (Fig 12). The scoring scheme allowed for clear differentiation between samples exhibiting strong, medium, and weak antibody responses (Fig 13). Quantitative assessment revealed a direct relationship between TiLV antigen concentration ranging from (40–0.625 ng) which is equivalent to 1.35 × 10¹¹ to 2.11 × 10⁹ copies µl^-1 and clump formation was observed up to four dilutions (5 ng; 1.69 × 10¹⁰) using a fixed TiLV-MAbs concentration (6.75 mg ml^-1) (Fig 14). Also decreased agglutination with dilution indicates the assay's potential for titer estimation, similar to ELISA. To validate the developed assay, a total 212 samples were tested and found mucus 73.53% (39/53), liver 77.35% (41/53), kidney 62.26% (33/53) and brain 67.92% (36/53); overall combine positive rate for TiLV-LAT was 70.28 (149/212) in O. niloticus.

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Fig 11. Schematic representation of the latex agglutination assay for TiLV antigen detection.

(A) Latex beads/particles are coated with TiLV-MAbs and incubated with tissue homogenate containing antigen (TiLV) against the coated antibody, which would induce agglutination of the latex beads. (B) Representative image of the agglutination assay using coated latex beads with TiLV-MAbs (segment 8).

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Fig 12. Agglutination of TiLV-MAbs latex beads in response to different concentrations of TiLV antigen with distinct agglutination scores (0-4).

The scores were assigned as 0 (or negative) ≤5% agglutination; 1 = 5-25% agglutination; 2 = 25-50% agglutination; 3 = 50-75% agglutination; 4 = 75-100% agglutination.

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Fig 13. The efficacy of the test for TiLV detection (n = 212) was determined semi-quantitatively by the aggregation score (1-4 denotes weak to strong antibody response) with respect to Fig 12.

Statistical analyses were performed using unpaired Student’s t-test (p values shown on graph).

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Fig 14. A. 2-fold serial dilution of PEG concentrated TiLV (40 ng) for quantitative identification. (B) Different samples were checked for specificity of TiLV-LAT, 1-TiPV, 2-NNV, 3- S. agalactiae and 4 -A. veronii.

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Performance of TiLV-LAT assay and the semi-nested RT-PCR test

To evaluate the performance of the TiLV-LAT assay, an analysis of 194 samples tested with semi-nested RT-PCR and TiLV-LAT revealed that semi-nested RT-PCR detected 151 positive samples, while TiLV-LAT detected 129 positive samples. Of these, 124 tested positive by both methods were considered true positives, and 5 were false positives. Furthermore, 43 samples were negative by semi-nested RT-PCR and 65 samples were found negative by TiLV-LAT assay. Similarly, 38 and 27 samples were considered true negative and false negative, respectively. The sensitivity and specificity of the TiLV-LAT assay were 82.12% and 88.37%, respectively. The percentage agreement between the assays was 83.51% (Table 3), while Cohen's kappa (κ) value of 0.596 demonstrated moderate agreement. A comprehensive statistical analysis of the TiLV-LAT assay is detailed in Table 4.

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Table 3. Confirmation of TiLV-LAT assay with the semi-nested RT-PCR test.

https://doi.org/10.1371/journal.pone.0344743.t003

Download:

Table 4. Statistical parameters of the TiLV-LAT test.

https://doi.org/10.1371/journal.pone.0344743.t004

Discussion

Tilapia, the third-most produced fish of all finfish species, was known for its hardiness and resilience to adverse environments and disease conditions in the past. However, the impact of intensive culture practices, other anthropogenic interventions, and climate change has resulted in several disease outbreaks in tilapia farming, leading to huge losses to the farmers. Diagnostics developed for TiLV can be listed under levels I-III, from presumptive to confirmatory lab-oriented and point-of-care methods targeting the pathogen as well as the effectors in the host. Aquaculture requires cost-effective and user-friendly diagnostic tools independent of the need for sophisticated laboratories and equipment. Non-lethal, non-invasive sampling methods enable effective disease screening [34] and/or monitoring in aquaculture systems without the need for euthanasia and animal sacrifice [43]; and to ensure a humane approach [44]. Therefore, we developed a field-deployable slide agglutination test for the detection of TiLV. In O. niloticus from various farms of Maharashtra state in India, aligning with the three Ds framework suggested by Louws et al. [45]. TiLV was isolated from infected tilapia and propagated in SSN-1 and OnlL cell lines. Plaque formation, cell elongation and detachment were observed in SSN-1 cells, whereas in OnlL, cellular vacuole formation, syncytium and monolayer destruction were prominent. TiLV-induced changes in cells are previously documented [22,34]. The infectious viral titre was assessed by TCID₅₀ in OnlL and SSN-1 cell lines. The results indicated that TiLV replicated more rapidly in SSN-1 cells with higher titers within 5–7 days post-infection [35], compared to OnlL cells, which were comparatively slower and took 10–12 days [22]. The TEM analysis of infected cells from various investigations confirmed the presence of ribonucleoprotein complexes with the envelope (~100 nm), the typical appearance of virus particles in the liver [15] and brain [35,46].

The segmented nature of the RNA genome of the virus likely contributes to its mutations, leading to inconsistent RT-PCR [47], so the confirmatory test requires multiple primer sets and multiple attempts targeting different segments of the viral genome to ensure accurate detection [21]. Diverse TiLV segments have been employed for PCR-based detection (segment 1 to segment 10) by various researchers [48]; segment-1, segment-5 and segment-9 [15]; segment-3 [21,23,24]; segment 8 [19]. In our study, we targeted segments 3 and 8 of the TiLV genome for RT-PCR testing. The RT-PCR (segment 3 and segment 8) test was used as a reference test for the newly developed slide agglutination test. The development of this test aimed to address issues such as laboratory intensiveness and inconsistency in TiLV RT-PCR tests. Here, we designed a serology-based field deployable slide test assay used at farmers and for hatcheries where TiLV testing is critical. A 15-mer synthetic peptide (KLH-CQ) was designed from the conserved sequences of all TiLV segment 8. The synthesized KLH-CQ peptide, mimicking the TiLV segment 8, was employed to produce monoclonal antibodies and a TiLV immunodiagnostic assay was developed. The antigen-antibody reactivity was validated by agglutination, a well-established serological technique used in blood typing and antibody detection [49–51]. Segment 8 protein is highly antigenic [19] and exhibits several characteristics, including hydrophobicity, positive charge, amphipathic nature, and a prominent alpha-helical structure (Table 2; Fig 2a-d). A core concept in biology is that the function of a protein is dictated by its structure. This principle is being harnessed to create small peptide mimetics, which mimic protein structures and utilize the parent molecules’ chemical properties [52]. Five hybridoma clones were selected for ELISA screening to confirm the reactivity and immunogenicity for TiLV detection. The cutoff for positive TiLV detection was established at an optical density of 1.24 ± 0.45. A highly reactive MAb clone was selected to develop a latex slide agglutination test out of the five clones. The developed anti-TiLV-MAbs were successfully adsorbed onto the surface of latex beads by the passive adsorption method [53] for developing the TiLV-latex agglutination test and named TiLV-LAT. The principle of the latex agglutination test is the formation of visible clumps when specific antibodies interact with the antigens.

The TiLV-LAT performance in this investigation revealed a sensitivity of 82.37% and a specificity of 88.37%, respectively. Due to the inverse relationship between sensitivity and specificity, maximizing sensitivity often requires sacrificing specificity [54]. The TiLV-LAT developed in the present study achieved lower sensitivity and higher specificity, which could result from the use of unpurified MAbs, i.e., clones without purification. The assays’ specificity and sensitivity were assessed through semi-nested RT-PCR. A comparison of TiLV-LAT assay with semi-nested RT-PCR revealed the higher sensitivity of semi-nested RT-PCR. While the overall agreement between TiLV-LAT and semi-nested RT-PCR was 83.12%, Cohen's kappa value of 0.596 suggests a moderate agreement [55].

The TiLV-LAT revealed a significant prevalence of TiLV infection in the farms, demonstrated by a high positive predictive value (PPV) of 96.12% and a comparatively low negative predictive value (NPV) of 58.46%. This pattern aligns with the principle that PPV increases and NPV decreases as disease prevalence rises [56]. Another parameter observed in the study was a positive likelihood ratio (LR+) of 7.06, which suggests a practical diagnostic test, as high LR+ values, achieved when specificity and sensitivity approach 1, provide strong positive evidence. Similarly, the negative likelihood ratio (LR-) of 0.2 indicates that a negative test is reliable for excluding the condition [56].

The latex agglutination assay demonstrates several characteristics that make it suitable for point-of-care (POC) testing [42]. It provides accurate and sensitive results, as evidenced by its 88.37% specificity and 82.12% sensitivity in our study. Further, the test is rapid, yielding results within three minutes. The test is user-friendly and requires no specialized equipment, as agglutination is visually detectable. The use of mucus as a test sample for detection using the TiLV-LAT reveals the property of its non-invasiveness in diagnosis [57]. Finally, its affordability can make it reasonable and widely accessible for TiLV testing. Test samples used for performing the assay were pooled samples of five fish each. Pooling tissue samples from 5 to 10 fish allows for the determination of TiLV infection status in ponds and farms and potentially for calculating infection prevalence Yamkasem et al. [58]. The newly developed TiLV-LAT assay showed the highest sensitivity in the liver at 77.35%, followed by mucus at 73.53%, the brain at 67.92%, and the lowest in the kidney at 62.26%. Though mucus showed 73.53% sensitivity, it can be a suitable sample for non-lethal detection of TiLV. TiLV, when present in the fish mucus, retains its infectivity, as evidenced by its ability to induce cytopathic effects on E-11 cells, suggesting that mucus can be used for effective screening [57]. Similarly, virus detection has been achieved in salmon through non-lethal sampling using mucus, faeces, and blood [59,60]. Antibody-based tests count on the specific affinity to the antigen targeted, and the level of precision of the binding is the key to a successful test. Antibody-based tests are extensively used in infectious diseases and aid in early and rapid diagnosis, which is suitable for mass screening. Our results highlight the potential use of latex agglutination test to detect TiLV antigen in various tissues. The segment 8 monoclonal antibody showed good efficiency in detection of the antigen. Further extensive validation of the test in field samples is highly desirable. To the best of our knowledge, this is the first report offering a non-lethal, dependable, farm-deployable approach to diagnose TiLV and is vital for tracking and limiting the virus spread.

Conclusion

Comprehensive methods were employed to detect the presence of TiLV in field samples, including RT-PCR, cell culture, and TEM, which present challenges in terms of time efficiency and consistency in the PCR analysis. However, robust synthetic peptide-based monoclonal antibodies were generated, and a latex agglutination test was optimized, which showed competitive efficiency compared to RT-PCR. Also TiLV-LAT test is more suitable than RT-PCR due to its simpler procedure, which avoids upstream and downstream processing. The developed assay can be field deployable that is crucial for the successful management and control of such diseases, especially in resource-constrained areas experiencing frequent outbreaks. It does not require specialized equipment such as thermal cyclers or microscopes and reduces the logistical burden, making the test suitable for use in remote locations. The latex agglutination assay in the current setup showed significant background signals when unpurified TiLV supernatant was used. This can be solved by processing the samples correctly. In future studies, it is recommended that the accuracy of the test be enhanced by measuring the clumping or macroscopic clusters using a microscope or other methods.

Supporting information

S1 Fig. Raw images of Polymerase Chain Reaction (PCR) agarose gel electrophoresis.

https://doi.org/10.1371/journal.pone.0344743.s002

(PDF)

S2 Fig. Graphical abstract.

https://doi.org/10.1371/journal.pone.0344743.s001

(TIF)

Acknowledgments

The authors extend their sincere gratitude to the Directors of ICAR-CIFE Mumbai, for their support in providing the essential facilities for this work.

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Figures

Abstract

Introduction

Materials and methods

Animal ethics statement

Designing, synthesis and purification of peptide

Immunization

Monoclonal antibody generation

Sample collection

RNA isolation and cDNA synthesis

RT-PCR detection

Virus isolation and identification

In vitro pathogenicity test and transmission electron microscopy (TEM)

Preparation of TiLV-MAbs coated latex particles

Latex agglutination assay for TiLV detection (TiLV-LAT assay)

Performance evaluation of TiLV-LAT assay against semi-nested RT-PCR

Statistical analysis

Results

Synthesis and structure analysis of peptide

Gross sign, PCR detection and isolation of TiLV

Monoclonal antibody production and characterization

TiLV detection by latex agglutination assay (TiLV-LAT assay)

Performance of TiLV-LAT assay and the semi-nested RT-PCR test

Discussion

Conclusion

Supporting information

S1 Fig. Raw images of Polymerase Chain Reaction (PCR) agarose gel electrophoresis.

S2 Fig. Graphical abstract.

Acknowledgments

References