Study the Acyclovir-resistant Strains of Herpes Simplex Virus

• Main Author: 詹淑楨
• Other Authors: 高全良
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/28528913768270064409

碩士 === 國立臺灣大學 === 醫事技術學研究所 === 86 === Herpes simplex virus is a double-strand DNA virus with envelope. Infections caused by HSV are usually asymptomatic or mild with mucocutaneous vesicles. More seriously, HSV can cause systemic infections, even death. The most safe and useful anti-HSV drug is Acyclovir. Since 1982, ACV-resistant HSV isolates have been reported. The number of resistant strain seems increasing. It is leads to therapy difficulties. In Taiwan area , there are few reports about the Acyclovir drug susceptibility of HSV. The screening of ACV resistant strains of HSV-1 has been done in our laboratory. But the situation about HSV-2 is not still clear. In order to understand the susceptibility of ACV to HSV-2 , 58 clinical isolates from Taipei V.D. center during period from 1992∼1993 and 17 clinical isolates from NTUH during period from 1997∼1998 were collected for study. The dye uptake and plaque reduction assay were used for analysis of drug sensitivity. The result revealed that the rate of ACV- resistant HSV was 1.17%(l/85) and its average of ID50 (50% inhibition dose) was l.3μg/ml. Comparing with HSV-1 (0.845μg/ml), it needs higher dose to inhibit the growth of HSV-2. We determined the thymidine kinase (TK) activity of plaque purification of the drug- resistant strain (930058). It was shown the TK activity of resistant strain was 48% of the sensitive strain. It is suggested that the strain (930058) was belonged TK-altered phenotype. The sequence of TK gene was analyzed. The 162 base at ATP binding site of TK gene was changed from G to A. It induced amino acid change from D to N. This is maybe caused the drug resistant appear. When screening drug resistant strains, we found some plaques of sensitive strains survive in higher dose (4μg/ml). Therefor, we isolate the plaques and assay the susceptibility of ACV. The strains of ID50 were ranged from 8μg/ml to 32μg/ml. The TK activity of these virus strains were only 3%∼10% of sensitive strains. They are TK-negative. For explore the mechanism of resistant strains, resistant strains compare TK gene sequence of these strains were compared. Results showed that 834372-S1 and 972823-S2 had 3G insertion ofTK sequence at position 617 , and it caused 207th amino acid changed and more a amino acid than sensitive strains. Frame shift by adding one G ofTK sequence at position 440 was found in one strain (942434-S2), and another frame shift by adding one c of TK sequence at position 1064 was found in one strain (973296-S4). There were one point mutation site of TK occurred in two strains (881506-S1, 961038-S1). We further compare growth curves in vitro, no obvious difference between resistant and sensitive strains. We cultured 3 sensitive strains which were purified in the medium with 0.5 μg/ml ACV in series. One of three strains had changed into resistant strain in third passage in Vero cells. The results indicated that ACV might caused direct induce mutation of virus which can process the emerging of resistant strains. It is needed further study to support the suggestion.