Studies on shrimp White Spot Syndrome Virus (WSSV): pathogenicity and characterization of WSSV genes that encode for ribonucleotide reductase subunits and a novel thymidine kinase-thymidylate kinase chimeric polypeptide

• Main Authors: Meng-Feng Tsai, 蔡孟峰
• Other Authors: Guang-Hsiung Kou
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/24854327957997284292

博士 === 國立臺灣大學 === 動物學研究所 === 89 === In the present study, we used PCR and in situ hybridization analysis to carry out long-term detection of white spot syndrome virus (WSSV) in the offspring of a WSSV-carrier brooder. The PCR screening results showed that WSSV can be carried in the offspring population at a low intensity for a very long time and that massive mortality never occurs. The detection rates of WSSV positive samples showed low levels from the egg to the PL1 stages, suggesting that the virus might not replicate well in the shrimp at early stages. In situ hybridization results of these WSSV lightly- infected specimens demonstrated higher WSSV tissue tropism in stomach, epidermis and gills, and only very rarely were WSSV-positive cells found in the lymphoid organ or other organs. We hypothesize that the absence of WSS outbreaks in this cultivated shrimp population was probably because the shrimp body defense mechanisms had managed to contain the virus under low-stress culture conditions. WSSV tk-tmk gene was identified from a 10.1kb WSSV genomic DNA fragment. This gene was predicted to encode a novel chimeric protein with 398 amino acids. Data from phylogenetic analysis and sequence alignment suggested that the gene may have resulted from the fusion of a cellular-type tk gene and a cellular-type tmk gene. Its unique arrangement may also provide a valuable gene marker for WSSV. Two WSSV genes which encode proteins with significant homology to the ribonucleotide reductase large (RR1) and small (RR2) subunits were identified. By 5’ RACE anaysis, the major rr1 transcript started at a position of —84 relative to the ATG translational start, while transcription of the rr2 gene started at nucleotide residue -68. A consensus motif containing the transcriptional start sites for rr1 and rr2 was observed (TCAc/tTC). Studies of the RR1 and RR2 phylogenic trees gave evidence that WSSV might be a new virus belonging to a new genus or family. The RR2 protein C-terminus is important for binding to the RR1 subunit. Many of the virus specific peptide inhibitors derived from the RR2 protein C-terminus have been developed to prevent the formation of the active RR1/RR2 tetramer. These peptide inhibitors can inhibit the virus RR activity without affecting the host RR activity We have cloned the rr2 gene of the Penaeus monodom to evaluate the possibility of a WSSV specific peptide inhibitor from the RR2 C-terminus. The P. monodom rr2 gene encoded a protein with 372 amino acids. The amino acid sequences of shrimp RR2 showed high homology to human and mouse RR2s (80% and 79%). A portion of the C- terminus in the P. monodom RR2 amino acids -FTLDADF- was the same as in mammalian RR2 proteins. However, WSSV RR2 protein C terminus amino acids were -YISYDDF-. This observation points up the possibility of developing specific peptide inhibitors against WSSV replication.