單純疱疹病毒第一型（Herpes simplex virus type 1, HSV-1）之感染容易在免疫功能低下病患中引起嚴重腦炎。治療單純疱疹病毒的最常見藥物為 Acyclovir（ACV），ACV 經由抑制病毒胸苷激酶（viral Thymidine Kinase, vTK）和病毒 DNA聚合酶（viral DNA polymerase, vDP）的活性以終止病毒的複製。然而，長時間接受 ACV 治療患者較易產生抗藥性之病毒株，導致罹病率和死亡率顯著增加。由於目前未有文獻報導南台灣地區 ACV 抗藥性 HSV-1 感染之盛行率與其突變基因的抗藥相關機制之探討。因此我們的研究旨在篩檢免疫功能低下患者中 ACV 抗藥性之 HSV-1 病毒，並聚焦於抗藥病毒株過去文獻尚未發表突變位點之抗藥性機制。
我們以 Plaque reduction assay 和 DNA reduction assay 篩檢成大醫院 294 位病人(其中免疫功能低下者為 222 例)的 420 病毒株之 ACV 抗藥性，具抗藥性之病毒株則再進一步以 Sanger 定序方法鑑定 vTK 和 vDP 基因全長之突變位點。結果顯示具 ACV 抗藥性 HSV-1 的病患比率為 1.7％ (5/294）與抗藥病毒 7 株，亦發現過去文獻尚未發表的基因突變位點有 4 個：其中一株的突變發生於 vTK 的 Y248D和在其他病毒株發現 vDP 中 683-688、R227H 和 A351D 之 3 個突變位點。
為了研究 vTK 的 Y248D 突變位點之抗藥性機制，我們利用 site-directed mutagenesis 技術製備具備單點突變的 vTK ( MtY248D-vTK)。後續把 WtKOS-vTK(wild-type vTK, ACV-sensitive type)和 MtY248D-vTK 送入 Vero 細胞組中過量表現。再以標準病毒株 KOS (sensitive strain)感染有加入 ACV 藥物之過量表現WtKOS-vTK 和 MtY248D-vTK 的 Vero 細胞中，實驗結果發現 WtKOS-vTK 組別病毒量顯著下降，而在 MtY248D-vTK 組別中發現 ACV 無法使 KOS virus 之病毒量減少。此外，以反相高效液相色譜(Reverse-phase high performance liquid
chromatography)方法證實在 MtY248D-vTK 組別無法偵測到 vTK 的磷酸化產物(ACV-triphosphate);以上結果顯示 MtY248D-vTK 具備使 ACV 失效之抗藥性能力。
總結，Y248D 突變可透過干擾 vTK 的磷酸化功能使 HSV-1 具 ACV 抗藥性。

Herpes simplex virus-1 (HSV-1) infections cause severe herpes encephalitis in immunocompromised patients. Acyclovir (ACV) as antiviral drug is often used for therapy of HSV-1 infections in patients. ACV could inhibit the activity of viral thymidine kinase (vTK) and viral DNA polymerase (vDP) for termination of HSV-1 replication. Prolonged treatment with ACV may result in the emergence of ACVresistant HSV-1, which is associated with significant morbidity and mortality. However, the occurrence rate of ACV-resistant HSV-1 infections in immunocompromised patients in Southern Taiwan is unclear and the mutation database remains limited. Therefore, our study aimed to understand the positive rate of ACV-resistant HSV-1 infections in immunocompromised patients and further study the ACV-resistant mechanism of the novel genetic mutations.
A total of 294 cases including 222 immunocompromised cases with 420 clinical isolates were screened for ACV-resistance by plaque reduction assay (PRA) and supplementary DNA reduction assay (DRA). ACV-resistant HSV-1 isolates were further sequenced by Sanger sequencing to identify the mutations in the full-length sequence of vTK and vDP. The study showed the positive rate of ACV-resistant HSV-1 patients was accounted for 1.7% (5/294) and 7 ACV-resistant HSV-1 isolates were found. Four unpublished mutations were from various ACV-resistant isolates including substitution Y248D in vTK and deletion in 683-688, R227H, and A351D of vDP.
To investigate the ACV-resistant mechanism of novel substitution Y248D, the mutant (MtY248D-vTK mimicked ACV-resistant strain) clone was prepared by sitedirected mutagenesis. WtKOS-vTK (wild-type vTK, ACV-sensitive type) and MtY248D-vTK was overexpressed in Vero cell line CCL-81 individually. Percent of plaque formation of reference HSV-1 strain KOS (standard sensitive type) decreased significantly in WtKOS-vTK group with ACV treatment. However, percentage of plaque formation of laboratory HSV-1 strain KOS showed no significantly difference between Vector group and MtY248D-vTK group with ACV. Moreover, compared to WtKOS-vTK group with ACV treatment, ACV-triphosphate as product for antiviral effect was not detected in MtY248D-vTK group by reverse phase-high performance liquid chromatography (RP-HPLC). It means ACV had no effect in KOS virus with overexpressing MtY248D-vTK. Overall, our data showed novel mutation Y248D could be contributed to HSV-1 ACV resistance by altering phosphorylation function of vTK.

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