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

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Author Biography

Pravata Kumar Pradhan, NBFGR, Lucknow

NBFGR, Lucknow

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