Abstract: The morphological evidence supports a key role for WDSV virus in the induction of skin tumours of fish. It is concluded that other biological and environmental factors mediate either expression of the tumour virus, tumour development or both. Abnormal and operculam growth of WDSV are always associated with fish tumors, which was noticed under experimental periods. In addition, mainly eyes and upper & lower jaw were recorded in middle days of experimental periods. Its clear positive indication of WDSV disorders. Irregular shape, colour, cloudiness, gas bubbles and small haemorrhagic lesions (red spots) also observed the study periods. The maximum and minimum cumulative mortality were recorded at tank A in 47th & 39th day of post inaugulation. Hence, WDSV less positive was found in 32nd day and positive bands were shown at 289bp respectively compared with 1kb DNA ladder. The moderate level was found in 39th days of experimental periods, it was shown at 479bp. Higher WDSV copies were recorded in 42nd day at 665bp, 479bp and 289bp respectively. Although the percentage of survival was noticed 100% where in 32nd day and minimum survival was observed 0 levels in ened of experimental periods (47th day). The RT-PCR detection and mortality & survival range of control tank B, were observed at 100% survival and mortality range is 0% level in experiment beginning onwards. It was shown negative house keeping gene band at 665bp, upto 47th day. The survival was noticed in minimum 0.0% and maximum was presented at 93.3%. The mortality ranges were occurred 13.3% in minimum and maximum 100% at day 32nd & 47st. The limit of detection was as severe, moderate and low infection pattern as 2000 copies/reaction (665 bp), 200 copies/reaction (479 bp) and 20 of copies/reaction (289 bp) was able to detect viral RNA from experimental animal tissues at levels ten-times lower than single tube PCR. Both higher WDSV infection and lower WDSV level infection has been amplified, and an in silico assay showed that WDSV of all genotypes can be amplified. WDSV was detected in target and non-target tissues of both diseased and asymptomatic fish. The negative control samples were showed only at 665bp, which was the product of housekeeping gene (internal control).The molecular weight marker was represented, 848 bp, 630 bp and 333 bp respectively. The lower level of infection were occurred in day 9 and severe level was found in 32 day onwards with 2000, 200 and 20 copies in under 665 bp, 479 bp and 289 bp respectively. A novel fluorescence-based staining method was developed for phosphoprotein analysis in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Similar to the Walleye dermal sarcoma virus (WDSV) protein plays an important role in determining host range but that other viral proteins are clearly required for full phosphoprotein pathogenicity to be manifest in concern species, Lates calcarifer appended complex as a fluoroprobe to selectively visualize phosphorylated proteins among total proteins. The pattern of phosphorylated and non-phosphorylated proteins were presented lower to high level in on under protein marker i.e., 25kDa – 200kDa. The degree of phosphorylation were obsereved 60 minutes in under the phosphatase treatment tank. The WDSV viral proein was separated under SDS-PAGE gel, using phosphoamino acid analysis. A comparison and identification specific phosphoprotein 64kDa band intensities of 86 kDa higher and 23 kDa lower phosphorylated protein were observed in under SDS-PAGE demonsattion that the pre antibody production system can achieve detection limits of WDSV infection level and comparable those different Kda proteins resolved on a polyacrylamide gel. Since the WDSV viral protein band intensity achieved with both chain bands varies for different viral specific proteins. This attempt is extends the detection capability of after antibody production with associated of immuno-purification of SDS specific (64 k Da phosphoprotein) gel band, standardization of viral proteins under ELISA and preparation of antibody against WDSV, using Rabbit for developing of antibody drug against WDSV.
Keywords: WDSV, RT-PCR, Lates calcarifer, experimental, mortality and SDS – PHAGE.
Title: Molecular detection and characterization of RNA tumour Walleye dermal sarcoma virus (WDSV) phosphoprotein in Sea bass Lates calcarifer by using nested PCR and SDS-PAGE
Author: S.Vaitheeswari, M.A.Badhul Haq, Chandan Tiwary
International Journal of Life Sciences Research
ISSN 2348-313X (Print), ISSN 2348-3148 (online)
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