The primary infection with herpes simplex virus type 1 (HSV-1) causes acute gingivostomatitis, which is usually benign in children. After the primary infection, HSV-1 establishes latent infection in the neuronal ganglia. HSV-1 latently infected in the neuronal ganglia reactivates from latency and causes skin infections such as herpes labialis. However, HSV-1 infections in immunocompromised patients are severe, intractable, and frequently recurrent. Chronic and intractable HSV-1 infections of immunocompromised patients require long-term acyclovir (ACV) administration, resulting in ACV-resistant (ACVr) HSV-1 infections in some patients. Most of the ACVr HSV-1 are due to mutations in the viral thymidine kinase (vTK), which is one of the target proteins for antiherpetic drugs. It was reported that vTK-deficient (vTK-) HSV-1 could establish latency in mouse trigeminal ganglia, but it could not reactivate from latency. The mechanism of the reactivation of TK- HSV-1 in humans is still unclear. The precise sensitivity assay of HSV-1 isolates to various vTK-associated drugs such as ACV, ganciclovir (GCV), and brivudine (BVdU) provides an useful information for proper treatment. A plaque reduction assay (PRA) is the most popular and the standard for an evaluation of the sensitivity of HSV-1 to antiviral drugs. HSV-1 is considered to be ACVr, if the 50% effective concentration (EC(50)) shows >2.0μg/ml. The PRA-based sensitivity system requires virus isolation, titration of the isolate, and PRA in the process of the assay, and thus takes a long period of time to obtain the results. This is a disadvantage in the treatment of HSV-1 infections in immunocompromised patients and in patients with severe forms of infection. Therefore, development of a rapid and reliable sensitivity assay system has been desired.

In the first chapter of this study, one of the mechanisms for the reactivation of TK- HSV-1 in humans was discussed by the clinical observation of and virological analyses on a child with congenital immunodeficiency for about 10 years from the primary infection at the age of 3 to the fatal infection, progressive multifocal encephalopathy, due to JC virus at the age of 13. The child suffered from a severe infection due to ACVr HSV-1 at the age of 8 (Age-8 ACVr infection). He suffered from recurrent ACV-sensitive (ACVs) and ACVr HSV-1 infections before and after this episode. HSV-1 isolates recovered from this patient were virologically analyzed. All the ACVr HSV-1 isolates were revealed to be TK- due to a single cytosine (C) deletion within the 4-C residues (positions 1061 to 1064) in the TK gene. Furthermore, TK-/ACVr HSV-1 with this mutation existed in the total populations of ACVs HSV-1 isolates recovered before and after the Age-8 ACVr infection, but not in that of ACVs HSV-1 isolate recovered at the primary infection. These results suggest that TK-/ACVr HSV-1 reactivated with that of TK+/ACVs HSV-1, and that TK-/ACVr HSV-1 might reactivate using the TK activity induced by the latently co-infected TK+/ACVs HSV-1 when it reactivated.

In the second chapter of this study, a novel drug-sensitivity assay system was developed. The 293T cells transiently expressed with vTK by transfection of the cells with the expression vector were infected with TK-/ACVr HSV-1 (HSV-1 TAR), and were then cultured in maintenance medium with or without designated concentration of ACV, GCV and BVdU. The replication of HSV-1 TAR was strongly inhibited by ACV, GCV and BVdU in 293T cells expressed with vTK of ACVs viruses, while it was not inhibited in those expressed with vTK of highly resistant and intermediately ACVr viruses. The inverse correlation was demonstrated between EC50s and inhibitory effect of these compounds on the replication of HSV-1 TAR. The HSV-1 isolates' solution recovered from 5 ACV-therapy resistant subjects with HSV-1 keratitis was sent from Chile in an inactivated form. Out of 5 isolates, one was demonstrated to be ACV, GCV, and BVdU-resistant and the rest were to be sensitive to these compounds. The time required for the measurement of the inhibitory effect could be minimized by the introduction of quantitative real-time PCR. The sensitivity of HSV-1 to antiviral drugs is usually determined by plaque reduction assay, which usually requires more than 10 days. However, the time required for the novel sensitivity assay system was less than 4 days, making it possible to treat patients with drug-resistant HSV-1 infections more properly.

In summary, the possible mechanism of the reactivation of the vTK-/ACVr HSV-1 in humans was discussed. Furthermore, a novel and rapid sensitivity assay system for HSV-1 to vTK-associated drugs was developed.

本研究は免疫不全症患者において問題となっている薬剤耐性単純ヘルペスウイルス1型(HSV-1)感染症に焦点を当て、ウイルス性チミジンリン酸化酵素欠損アシクロビル耐性(vTK- / ACVr) HSV-1の再活性化機構の解明および従来法に優る新規迅速薬剤感受性試験法の開発を試みたものであり、下記の結果を得ている。

1.ウィスコットアルドリッチ症候群(WAS)患児の10年に及ぶ観察とウイルス分離を通して、初感染時には分離されなかったvTK- / ACVr HSV-1TAR (vTK遺伝子の1061から1064番目の4-C残基にCが挿入)が最初のACV耐性株から支配的に分離されたことが示された。

2.WAS患児より初感染以降、薬剤感受性株として分離された株も含め、再活性化時に常にTARが分離されたことが示された。

3.1.および2.からTARが感受性株のvTK活性を利用して再活性化する可能性が示された。つまり、マウスモデルでは再活性化が認められないvTK-耐性株が、ヒトにおいては感受性株との共感染によって再活性化される可能性が示された。

4.下記のコンセプトによって新規迅速薬剤感受性試験法が構築されたことが示された。検査対象のウイルス由来のvTK遺伝子をpcDNA3.1へ搭載し、293T細胞へトランスフェクションさせ、TARをサロゲートウイルスとして感染後ACV存在下で培養をすると、検査対象ウイルスの薬剤感受性に対応してTARの増殖が変化することが示された。また、ACV 40μg/mlにおける定量的リアルタイムPCRによるウイルスDNA量と各ウイルスのEC(50)との間には強い正の相関が確認された。一連の操作は4日以内で遂行可能であり、10日を超える従来のプラーク減少法と比べて著しく迅速であることが示された。

5.4.において示された結果は、ACV以外のTK関連薬剤であるガンシクロビルやブリブジンにおいても同様の相関が示された。

6.新規迅速薬剤感受性試験法は、DNAポリメラーゼ変異由来の薬剤耐性株を検出することはできないが、薬剤耐性株全体の95%以上を占めるvTK変異に比べてその占有率は極めて低いため大きな問題とはならないことが示された。

7.海外からの臨床分離株への新規迅速薬剤感受性試験法の応用から、ろ紙へ滴下した患者サンプルだけで本法が実施可能であることが示されると同時に、国際的な検査依頼に容易に応じることができることが示された。

以上、本論文はWAS患児の長期間におよぶウイルス解析によってvTK- / ACVr HSV-1の免疫不全症患者における再活性化機構を明らかにした。また、新しいコンセプトのもと迅速薬剤感受性試験法が開発された。本研究は、薬剤耐性ヘルペスウイルス感染症の病態の理解と診断法の確立に重要な貢献を成すと考えられ、学位の授与に値するものと考えられる。