人巨細胞病毒耐藥突變及其檢測的研究進展

夏長勝,張 正

(北京大學人民醫院檢驗科,北京100044)

HCMV屬于皰疹病毒科,β皰疹病毒亞科,巨細胞病毒屬,人皰疹病毒5型。HCMV基因組為線性雙股DNA分子,長約230 kb,由長單一序列(UL)和短單一序列(US)構成[1,2]。HCMV在自然界廣泛存在,正常人群中自然感染普遍。大多數感染者無明顯癥狀,但在嬰幼兒和免疫功能受抑制的個體可引起嚴重疾病。目前常用的抗HCMV藥物主要有更昔洛韋(GCV)、磷甲酸鈉(FOS)和西多福韋(CDV)等。免疫抑制患者需長期進行抗病毒治療,使得巨細胞病毒的耐藥成為一個嚴重的問題。臨床耐藥株大多分離自HCMV感染久治不愈患者的標本[3]。抗病毒藥物有毒副作用,如GCV可使中性粒細胞減少,易引發致命性條件感染,而FOS和CDV則有嚴重的腎毒性。因此,臨床在長期使用抗HCMV藥物治療時需監測HCMV的耐藥性,以便指導臨床制定合理的治療方案。本文就人巨細胞病毒耐藥的分子機制和耐藥突變的檢測方法進行綜述。

1 人巨細胞病毒耐藥突變的分子機制

1.1 HCMV UL97基因耐藥突變

UL97基因全長2124個堿基,編碼707個氨基酸組成的磷酸轉移酶[1],其基序具有蛋白激酶特征,并且和其它皰疹病毒的激酶基因具有同源性。GCV是第一個被開發及應用最普遍的治療HCMV感染的高效藥物,它的選擇性抗病毒活性依賴于UL97激酶對其進行初始磷酸化。因此UL97基因突變可以引起HCMV對GCV發生耐藥。臨床絕大多數的GCV耐藥株是由于UL97基因突變。UL97基因多個位點突變均可發生GCV的耐藥,具體如圖1[4](A)及表1所示[5-18]。臨床分離的HCMV GCV耐藥株是由于UL97基因的460位、520位及590位到607位密碼子發生突變引起,絕大多數為發生了M460V、M460I、A594V和 L595S突變所致,其分別占臨床GCV耐藥分離株的14%、9%、29%和23%[19]。

1.2 HCMV UL54基因耐藥突變

UL54基因全長3729個堿基,編碼由1242個氨基酸組成的DNA聚合酶[1]。GCV、FOS和CDV均可作為UL54的替代底物發揮抗病毒作用。UL54基因耐藥突變可表現為耐GCV、CDV及FOS[20-26],其突變位于高度保守區,如圖1(B)及表1所示。UL54耐GCV突變大多同時交叉耐CDV[21,22]。有意思的是這種耐藥突變主要是由于長期使用GCV篩選獲得,而非使用CDV獲得。

圖1 UL97和UL54基因突變圖解

2 人巨細胞病毒耐藥的檢測

2.1 表型檢測

表型檢測首先需分離培養病毒,然后在系列濃度藥物的細胞培養中檢測病毒的復制情況。表型檢測中的空斑減少試驗(PRA)是評價HCMV耐藥性的金標法[27]。PRA即病毒在系列濃度藥物的培養基中生長7-10天,然后根據各藥物濃度下的病毒蝕斑數來確定藥物的半數抑制濃度(IC50)。此方法獲得實驗結果周期較長超過1個月。表型檢測除了可檢測病毒的蝕斑數,還可通過利用PCR檢測病毒DNA、免疫組化檢測HCMV pp65抗原表達、流式細胞術檢測病毒感染細胞[28]以及報告細胞株檢測[29]等方法來確定病毒的耐藥性。這些方法可縮短檢測周期,但是由于均需要進行病毒培養,從而限制了表型檢測在臨床上的應用。表現檢測中的重組表型檢測是確定臨床分離株中發現新的基因突變是否引起耐藥的參考方法。重組表型檢測,即通過同源重組將突變序列轉移到參考病毒株上,再進行耐藥表型檢測。目前技術發展到可以利用獨特的限制性內切酶位點進行靶基因位點特異性重組[8],運用報告基因進行快速病毒定量[30]。克隆HCMV全基因組的細菌人工染色體被構建為含UL97的變異體,利用此系統也可以進行重組表型檢測[31,32]。表型檢測可以確定未知突變能否引起耐藥性改變,但表型檢測由于需要進行病毒培養,存在如下缺點:①耗時費力;②缺乏統一標準;③室內誤差較大。HCMV的耐藥表型檢測主要運用于研究型實驗室,而在臨床實驗室的應用有限。

表1 HCMV相關的耐藥突變

2.2 基因型檢測

基因型檢測方法主要包括PCR限制性片段長度多態性分析(PCR RFLP)、PCR直接測序法、熒光標記雜交雙探針PCR溶解曲線法(FH-PCR-MC)、分子信標PCR溶解曲線法和擴增阻礙突變系統實時PCR法(ARMS RT PCR)等。PCR RFLP方法是根據已知HCMV基因組中與藥物抗性有關DNA序列的突變可丟失或創造一個限制性內切酶位點,先進行PCR再利用內切酶酶切分析[5,12,33]。有時基因突變沒有改變限制性內切酶的位點時,可通過引物設計引入一個限制性內切酶位點。PCR RFLP檢測方法優點是較快速(2天出結果)、敏感(突變株達到10%即可檢測到),且不需培養病毒(可直接檢測標本);其缺點是只能檢測已知的耐藥突變,對于未知可能引起的耐藥突變無法檢測,對于突變株感染和突變株野生株混合感染往往難以區分。此方法依靠凝膠電泳判斷結果,敏感性較差,假陰性率較高。由于需要取PCR產物進行酶切及電泳,易造成PCR實驗室污染,所以其使用受到一定的限制。直接測序法是指對HCMV的UL97基因或UL54基因進行PCR擴增,再對PCR擴增產物進行測序,通過與參考病毒株DNA序列的比對識別突變位點[28,34-36]。一般認為測序法是較為客觀的方法,獲得的信息較全面。隨著測序服務的專業化和商品化,測序結果變得準確可靠,測序費用變得便宜,因此直接測序法檢測HCMV耐藥突變具有較高的應用價值。但PCR產物直接測序對感染病毒中優勢株的檢測效果較好,較難發現非優勢株,對混合株的感染檢出受限,從而限制了其臨床應用。報道顯示其僅能檢測突變超過25%的突變病毒株[37]。FH-PCR-MC法是基于雜交雙探針技術和熒光共振能量轉移(FRET)原理,結合融解曲線分析的一種突變檢測方法。雜交雙探針熒光PCR完成后,以恒定的變溫速率緩慢地升高擴增產物的溫度,可測定PCR產物的解鏈溫度(Tm值)。由于野生株和變異株的解鏈溫度不同,借此可識別。G?hring等[38-39]利用此種方法成功檢測HCMV UL97基因多個密碼子的耐藥突變。該方法能實時監測PCR后解鏈過程中熒光信號的變化,操作簡便,特異性和準確性較好,能區分野生型、不同變異類型和混合型,但相對敏感性略低,不能對變異株在病毒群體中的比例進行定量,且容易出現假陽性結果。分子信標PCR溶解曲線法和FH-PCR-MC法檢測突變的原理相似,不同的是利用分子信標替代了雙探針。分子信標不像雙探針,由于其莖部不能和模板完全配對結合,因此穩定性較差。Yeo等[40]成功建立了檢測UL97基因460密碼子突變的分子信標PCR溶解曲線法。除了上述方法,本實驗室建立了ARMS PCR結合SYBR GreenⅠ的實時PCR法[41]。實時ARMS PCR方法的關鍵技術在于引物的設計。一般檢測單個位點突變需設計3條引物,1條為公共引物,1條引物為針對突變模板的突變引物,另外一條為針對混合模板的引物。公共引物結合突變引物要求能有效擴增突變模板,對于野生模板不擴增或低效擴增;公共引物結合針對混合模板的引物則可以同時擴增突變和野生模板。ARMS PCR引物設計一般采用引物3'端最后一個堿基錯配可引起產物銳減的原理進行設計。但僅一個堿基錯配往往不能有效阻止野生株的擴增,此時需要在引物3'端倒數第二個堿基上引入額外的錯配堿基才可能達到有效阻止野生株的擴增。ARMS PCR需要試驗驗證不同突變引物的效果,然后篩選最佳的突變引物[42,43]。ARMS實時PCR則是利用SYBR GreenⅠ、TaqMan探針或分子信標等進行實時監測。SYBR GreenⅠARMS PCR方法簡單、費用低廉,但其易受非特異性擴增產物和引物二聚體的干擾,特異性不高。運用TaqMan探針或分子信標的ARMS實時PCR則排除非特異性擴增和引物二聚體的影響,采用突變引物及針對混合模板的引物、公共引物和公共探針或公共分子信標可對病毒突變株占病毒總量的百分比進行檢測,并可對突變株進行準確定量[44,45]。總之,基因型檢測不需病毒培養,可直接采用臨床標本提取DNA檢測,比較簡便快捷。但基因型檢測方法除了直接測序法,其它方法均只能檢測已知位點的耐藥突變,對于未知的突變信息無法了解。

隨著器官、骨髓移植術的開展以及艾滋病的流行等,免疫受抑個體將不斷增加,HCMV感染者會越來越多。抗病毒藥物的廣泛使用將使得HCMV的耐藥性變成嚴重的問題。臨床必須不斷了解HCMV耐藥機制和檢測方法,從而監測HCMV的耐藥性和制定治療方案。研究型實驗室可以通過表型檢測方法確定新的突變是否引起耐藥;普通臨床實驗室則可利用基因型檢測方法檢測已知的耐藥突變,或是直接測序法了解更多的突變信息。

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