Development of a Competitive Chain Reaction Assay For Quantitative Analysis of White Spot Syndrome Virus Gene Transcription and Viral Replication in Shrimps
Tan, Lee Tung (2005) Development of a Competitive Chain Reaction Assay For Quantitative Analysis of White Spot Syndrome Virus Gene Transcription and Viral Replication in Shrimps. PhD thesis, Universiti Putra Malaysia.
Despites much research on infectivity and diagnostics of white spot syndrome virus (WSSV), little is known about the viral replication kinetics and quantitative gene expressions. Therefore, a time course quantitative study was carried out using competitive polymerase chain reaction (cPCR) to measure viral growth in grow-out Penaeus monodon experimentally infected via feeding of WSSV infected tissue. The current tissue tropism studies demonstrated that gills have higher viral load followed by integument and abdominal muscle. Gills and integument were infected as early as 14 hour post infection (hr p.i.) compared to 24 hr pi. for abdominal muscle. Gills are therefore recommended for extraction of DNA in routine PCR screening of WSSV. A classification of infection level was proposed to categorise infection into light (0 to 24 hr p.i.), moderate (24 to 48 hr p.i.) and moribund (48 to 120 hr pi.) stage according to viral loads detected in gills, which were 0 to 1 x lo3, 1 x lo3 to 1 x 10' and 1 x 10' to 1 x lo9 copies per mg tissue respectively for the three infection stages. As the viral load was low at light infection, but increased exponentially at moderate infection and maintained at high level at moribund infection, such pattern of growth in viral loads is comparable to the eclipse, logarithmic and plateau phase of viral growth curve. White spots and reddish discoloration on the exoskeleton were apparent in moderate and moribund infection stage, but terminal clinical signs such as abnormal swimming behaviour and heavy mortality could only be observed in the later. Previous studies on WSSV early genes expression were often qualitative rather than quantitative. By using competitive reverse transcriptase PCR (cRT-PCR), early gene ribonucleotide reductase large subunit (RR1) and thymidine kinasethymidylate kinase (TK-TMK) mRNA expressions were non detectable at light infection stage (12 hr p.i.), but abundant at moderate (24 hr p.i.) and moribund (60 hr p.i. and above) infection stages. Geomeans of RR1 expression in whole heart samples were 9.69 x lo4 and 2.36 x 10' copies at moderate and moribund infection stage respectively. Thus, both genes are probably vital in establishing WSSV infection, and their expressions are useful as marker in anti-viral studies of WSSV.Shrimp immunity was emphasised under the European Commission's Shrimp Immunity and Disease Control (SI & DC) project. At present, prophenoloxidase (proPo) activating system and penaeidins, the predominant antimicrobial peptides, are well studied in bacterial and fungal infection, but not in viral infection. The mRNA expression of prop0 was detected low and infrequent throughout infection with two-step PCR in heart and lymphoid organ. Penaeidin expression was however abundant with geomean of 4.35 x lo4 copies in light infection (12 hr p.i.) but downregulated to 8.94 x lo3 copies at moderate infection (24 hr p.i.) and non-detectable at moribund stage in whole heart samples. The lack of penaeidin and prop0 mRNA upregulation suggests that they have little if any importance in the response to viral infection.
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