Table 1.
Primers used in the current study.
Table 2.
Physicochemical parameters of KLH-CQ peptide in inwards like environment.
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.
Fig 2.
Secondary structure analysis of novel KLH-CQ peptide using PSI-PRED (A) and GOR4 (B) online tools.
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.
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.
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).
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).
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.
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.
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).
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.
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).
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.
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).
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.
Table 3.
Confirmation of TiLV-LAT assay with the semi-nested RT-PCR test.
Table 4.
Statistical parameters of the TiLV-LAT test.