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研究生：	郭昭佑 Kuo, Jhao-You
論文名稱：	免疫功能低下病人之單純疱疹病毒抗藥性篩檢與胸苷激酶上突變Y248D之探討 Drug Resistant Screening and Investigation of Y248D Mutation on Thymidine Kinase among Immunocompromised Patients with HSV-1 infections
指導教授：	蔡慧頻 Tsai, Huey-Pin
學位類別：	碩士 Master
系所名稱：	醫學院 - 醫學檢驗生物技術學系 Department of Medical Laboratory Science and Biotechnology
論文出版年：	2021
畢業學年度：	109
語文別：	英文
論文頁數：	68
中文關鍵詞：	單純疱疹病毒第一型 (HSV-1) 、Acyclovir（ACV) 、病毒胸苷激酶(vTK）、ACV抗藥性 (ACV-resistant) 、反相高效液相色譜 (Reverse-phase high performance liquid chromatography）
外文關鍵詞：	Herpes simplex virus-1 (HSV-1), Acyclovir (ACV), Viral thymidine kinase (vTK), ACV-resistant, Reverse-phase high performance liquid chromatography (RP-HPLC)
相關次數：	點閱：109 下載：0
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中文摘要	I
Abstract	III
致謝	V
Contents	VI
Ⅰ. Abbreviation      1
Ⅱ. Introduction      2
1. Introduction of Herpes simplex virus type 1 (HSV-1)      2
1.1 Classification, structure, and life cycle of HSV-1      2
1.2 Clinical manifestations of HSV-1 infection      5
1.3 Long-term latency and recurrence in trigeminal ganglia      5
2. Antiviral agents for HSV-1 infection      5
3. ACV-resistant HSV-1      6
3.1 Positive rate of ACV-resistant HSV-1 in immunocompetent and immunocompromised patients      6
3.2 Mechanism of resistance to ACV      7
3.3 Laboratory assays for detection of ACV-resistant HSV-1      8
4. Research motive      9
4.1 Significance      9
4.2 Specific Aims      9
Ⅲ. Materials and methods      10
1. Clinical specimens and laboratory HSV-1 strains      10
2. Cell culture and virus isolates      10
3. Antiviral compound      11
4. Phenotypic analysis      11
5. Genotypic analysis      12
6. Overexpression of plasmid pAL119-TK in Vero cell CCL-81      13
7. Western blotting      17
8. Reverse phase high performance liquid chromatography (RP-HPLC)      17
9. Protocol of growth curve test      19
Ⅳ. Results      21
1. Clinical manifestations of HSV-1 infected patients in NCKUH      21
2. Phenotypic analysis of ACV-resistant (ACV-R) HSV-1 clinical isolates      21
3. Genotypic analysis of ACV-resistant HSV-1 clinical isolates      22
4. ACV-resistant HSV-1 single clone from clinical isolate by plaque purification	 
      24
5. Expression of vTK in ACV-resistant HSV-1 isolate with the novel mutation Y248D was significantly lower than in ACV-sensitive HSV-1 isolate      25
6. In vitro model was constructed for overexpressing viral thymidine kinase      25
7. Validation the ACV resistance effect of novel mutation Y248D      26
8. Mutation Y248D does not interfere with the viral replication rate      29
Ⅴ. Discussion.      30
Ⅵ. References      35
Ⅶ. Tables and Figures      39
Table 1. Immunocompromised patients with HSV-1 infection from January 2014 to July 2019 (n=222)      39
Table 2. Phenotypic and genotypic analysis of drug resistant of HSV-1 laboratory reference strains      40
Table 3. Phenotypic and genotypic analysis of ACV resistant among clinical isolates from HSV-1 infected patients      41
Table 4. Phenotypic analysis of antiviral activity of FOS and CDV among clinical isolates from ACV-resistant HSV-1 patients      42
Figure 1. Plaque reduction assay of clinical HSV-1 isolates with CDV      43
Figure 2. Plaque reduction assay of clinical HSV-1 isolates with FOS      44
Figure 3. Phenotypic and genotypic analysis of unpurified clinical isolates C118-1, C118-3, and C118-4 with ACV from patient B      45
Figure 4. Plaque reduction assay of purified and unpurified HSV-1 isolates from patient B with ACV      46
Figure 5. Genotypic analysis of purified clinical isolates C118-1, C118-3, and C118-4 with ACV      47
Figure 6. HSV-1 vTK expression of laboratory strains and clinical HSV-1 isolates by western blot      48
Figure 7. Construction of plasmids overexpressing in vitro wildtype and mutant viral thymidine kinase      49
Figure 8. Overexpressing various in vitro viral thymidine kinase in Vero cell      50
Figure 9. Overexpressing various in vitro viral thymidine kinase in 143B cell      51
Figure 10. Plaque formation percentage of ACV-R-HSV-1 (purified isolates) in Vero cell overexpressing in vitro vTK      52
Figure 11. Plaque formation percentage of ACV-S-HSV-1 (KOS and purified isolate) in Vero cells overexpressing in vitro vTK      53
Figure 12. RP-HPLC/UV analysis of standard ACVTP (ACV-triphosphate)      54
Figure 13. RP-HPLC/UV analysis and calibration curve of standard ACVTP after extracted by perchloric acid      55
Figure 14. RP-HPLC/UV analysis of ACVTP (RT: 5.3) in different groups overexpressing in vitro vTK      56
Figure 15. RP-HPLC/UV analysis of ACVTP (RT: 5.1) in different groups overexpressing in vitro vTK in 143B cell (Vector / WtKOS-vTK / MtY248D-vTK group)      57
Figure 16. Calibration curve of the extracted ACVMP (RT: 2.4)      58
Figure 17. RP-HPLC/UV analysis of ACVMP (RT: 2.4) in 143B cell group overexpressing in vitro vTK (WtKOS-vTK group)      59
Figure 18. RP-HPLC/UV analysis of ACVMP (RT: 2.4) in different 143B cell groups overexpressing in vitro vTK (Mock / Vector / WtKOS-vTK group)     60
Figure 19. RP-HPLC/UV analysis of ACVMP (RT: 2.4) in different groups overexpressing in vitro vTK (Mock / Vector / WtKOS-vTK / MtY248D-vTK group)	61
Figure 20. Cytopathic effect of laboratory strains and clinical HSV-1 isolates observed at 6 / 12 / 24 / 36 / 48 / 72 hours by inverted microscope      62
Figure 21. Growth curve of laboratory strains and clinical HSV-1 isolates      63
Ⅷ. Applendix      64
Table S1. Primers, probes, and PCR conditions for DNA reduction assay      64
Table S2. Primers for UL23 and UL30 gene sequence amplification and analysis	65
Figure S1. HSV-1 genome structure      66
Figure S2. Gene analysis and protein prediction of viral thymidine kinase      67
Figure S3. Full sequence map for plasmid pAL119-TK (https://www.addgene.org/21911/)      68
                                    

1 Bedadala, G., Chen, F., Figliozzi, R., Balish, M. & Hsia, V. et al. Construction and Characterization of Recombinant HSV-1 Expressing Early Growth Response-1. ISRN Virol. 2014;2014:629641. (2014).
2 Bestman-Smith, J. & Boivin, G et al. Drug resistance patterns of recombinant herpes simplex virus DNA polymerase mutants generated with a set of overlapping cosmids and plasmids. J Virol 77, 7820-7829 (2003).
3 Blaho, J. A., Morton, E. R. & Yedowitz, J. C. et al. Herpes simplex virus: propagation, quantification, and storage. Curr Protoc Microbiol Chapter 14, Unit 14E 11 (2005).
4 Brunnemann, A. K. et al. Relevance of non-synonymous thymidine kinase mutations for antiviral resistance of recombinant herpes simplex virus type 2 strains. Antiviral Res 152, 53-57 (2018).
5 Burrel, S. et al. Surveillance of herpes simplex virus resistance to antivirals: a 4-year survey. Antiviral Res 100, 365-372 (2013).
6 Burrel, S. et al. Phenotypic and genotypic characterization of acyclovir-resistant corneal HSV-1 isolates from immunocompetent patients with recurrent herpetic keratitis. J Clin Virol 58, 321-324 (2013).
7 Burrel, S., Deback, C., Agut, H. & Boutolleau, D. et al. Genotypic characterization of UL23 thymidine kinase and UL30 DNA polymerase of clinical isolates of herpes simplex virus: natural polymorphism and mutations associated with resistance to antivirals. Antimicrob Agents Chemother 54, 4833-4842 (2010).
8 Chen, S. H. et al. Failure of thymidine kinase-negative herpes simplex virus to reactivate from latency following efficient establishment. Journal of virology, 78(1), 520–523. (2003)
9 Frobert, E. et al. Resistance of herpes simplex viruses to acyclovir: an update from a ten-year survey in France. Antiviral Res 111, 36-41 (2014).
10 Frobert, E. et al. Herpes simplex virus thymidine kinase mutations associated with resistance to acyclovir: a site-directed mutagenesis study. Antimicrob Agents Chemother 49, 1055-1059 (2005).
11 Fujii, H. et al. Application of next-generation sequencing to detect acyclovir-resistant herpes simplex virus type 1 variants at low frequency in thymidine kinase gene of the isolates recovered from patients with hematopoietic stem cell transplantation. J Virol Methods 251, 123-128 (2018).
12 Hussin, A., Md Nor, N. S. & Ibrahim, N. et al. Phenotypic and genotypic characterization of induced acyclovir-resistant clinical isolates of herpes simplex virus type 1. Antiviral Res 100, 306-313 (2013).
13 Ibanez, F. J. et al. Experimental Dissection of the Lytic Replication Cycles of Herpes Simplex Viruses in vitro. Front Microbiol 9, 2406 (2018).
14 Jee Hyun Kim. et al. Establishment and use of a cell line expressing HSV-1 thymidine kinase to characterize viral thymidine kinase-dependent drug-resistance. Antiviral Res 54, 163-172 (2002)
15 Jiang, Y. C., Feng, H., Lin, Y. C. & Guo, X. R. et al. New strategies against drug resistance to herpes simplex virus. Int J Oral Sci 8, 1-6 (2016).
16 Kakiuchi, S. et al. Association of the Emergence of Acyclovir-Resistant Herpes Simplex Virus Type 1 With Prognosis in Hematopoietic Stem Cell Transplantation Patients. J Infect Dis 215, 865-873 (2017).
17 Kaspar, M. et al. Stepwise characterization of non-synonymous mutations in the HSV-1 thymidine kinase gene by different functional assays. J Virol Methods 247, 51-57 (2017).
18 Kukhanova, M. K., Korovina, A. N. & Kochetkov, S. N. et al. Human herpes simplex virus: life cycle and development of inhibitors. Biochemistry (Mosc) 79, 1635-1652 (2014).
19 Leary, J. J., Wittrock, R., Sarisky, R. T., Weinberg, A. & Levin, M. J. et al. Susceptibilities of herpes simplex viruses to penciclovir and acyclovir in eight cell lines. Antimicrob Agents Chemother 46, 762-768 (2002).
20 McGeoch, D. J., Rixon, F. J. & Davison, A. J. et al. Topics in herpesvirus genomics and evolution. Virus Res 117, 90-104 (2006).
21 Mettenleiter, T. C., Klupp, B. G. & Granzow, H. et al. Herpesvirus assembly: a tale of two membranes. Curr Opin Microbiol 9, 423-429 (2006).
22 Michael Gebelein. et al. Nucleotide preparation from cells and determination of nucleotides by ion-pair high-performance liquid chromatography. Journal of chromatography 577, 146-150 (1992)
23 Monjo, A. L. et al. Photodynamic Inactivation of Herpes Simplex Viruses. Viruses 10, 532 (2018).
24 Pan, D. & Coen, D. M. et al. Quantification and analysis of thymidine kinase expression from acyclovir-resistant G-string insertion and deletion mutants in herpes simplex virus-infected cells. J Virol 86, 4518-4526 (2012).
25 Piret, J. & Boivin, G. et al. Resistance of herpes simplex viruses to nucleoside analogues: mechanisms, prevalence, and management. Antimicrob Agents Chemother 55, 459-472 (2011).
26 Piret, J. & Boivin, G. et al. Antiviral drug resistance in herpesviruses other than cytomegalovirus. Rev Med Virol 24, 186-218 (2014).
27 Piret, J. & Boivin, G. et al. Antiviral resistance in herpes simplex virus and varicella-zoster virus infections: diagnosis and management. Curr Opin Infect Dis 29, 654-662 (2016).
28 Piret, J. et al. Contrasting effects of W781V and W780V mutations in helix N of herpes simplex virus 1 and human cytomegalovirus DNA polymerases on antiviral drug susceptibility. J Virol 89, 4636-4644 (2015).
29 Rabelo, V. W., Romeiro, N. C., Paixao, I. & Abreu, P. A. et al. Mechanism of resistance to acyclovir in thymidine kinase mutants from Herpes simplex virus type 1: a computational approach. J Biomol Struct Dyn 38, 2116-2127 (2020).
30 Renner, D. W. & Szpara, M. L. et al. Impacts of Genome-Wide Analyses on Our Understanding of Human Herpesvirus Diversity and Evolution. J Virol 92 (2018).
31 Sauerbrei, A., Vodisch, S., Bohn, K., Schacke, M. & Gronowitz, S. et al. Screening of herpes simplex virus type 1 isolates for acyclovir resistance using DiviTum(R) assay. J Virol Methods 188, 70-72 (2013).
32 Schubert, A. et al. Single nucleotide polymorphisms of thymidine kinase and DNA polymerase genes in clinical herpes simplex virus type 1 isolates associated with different resistance phenotypes. Antiviral Res 107, 16-22 (2014).
33 Tanaka, M., Kagawa, H., Yamanashi, Y., Sata, T. & Kawaguchi, Y. et al. Construction of an excisable bacterial artificial chromosome containing a full-length infectious clone of herpes simplex virus type 1: viruses reconstituted from the clone exhibit wild-type properties in vitro and in vivo. J Virol 77, 1382-1391 (2003).
34 Tardif, K. D., Jorgensen, S., Langer, J., Prichard, M. & Schlaberg, R. et al. Simultaneous titration and phenotypic antiviral drug susceptibility testing for herpes simplex virus 1 and 2. J Clin Virol 61, 382-386 (2014).
35 Tenser RB. et al. Role of herpes simplex virus thymidine kinase expression in viral pathogenesis and latency. Intervirology. 1991;32(2):76-92. (1991)
36 Thi, T. N. et al. Rapid determination of antiviral drug susceptibility of herpes simplex virus types 1 and 2 by real-time PCR. Antiviral Res 69, 152-157 (2006).
37 Tischer, B. K., von Einem, J., Kaufer, B. & Osterrieder, N. et al. Two-step red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli. Biotechniques 40, 191-197 (2006).
38 Van der Beek, M. T. et al. Rapid susceptibility testing for herpes simplex virus type 1 using real-time PCR. J Clin Virol 56, 19-24 (2013).
39 Vikas, R. et al. HSV susceptibility to acyclovir - genotypic and phenotypic characterization. Antivir Ther 24, 141-145 (2019)

校內：2026-08-20公開
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