夏長勝,張 正

(北京大學(xué)人民醫(yī)院檢驗科,北京100044)

HCMV屬于皰疹病毒科,β皰疹病毒亞科,巨細(xì)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(gòu)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(shù)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(yán)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(jìn)行抗病毒治療,使得巨細(xì)胞病毒的耐藥成為一個嚴(yán)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(biāo)本[3]。抗病毒藥物有毒副作用,如GCV可使中性粒細(xì)胞減少,易引發(fā)致命性條件感染,而FOS和CDV則有嚴(yán)重的腎毒性。因此,臨床在長期使用抗HCMV藥物治療時需監(jiān)測HCMV的耐藥性,以便指導(dǎo)臨床制定合理的治療方案。本文就人巨細(xì)胞病毒耐藥的分子機(jī)制和耐藥突變的檢測方法進(jìn)行綜述。

1 人巨細(xì)胞病毒耐藥突變的分子機(jī)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(zhuǎn)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(fā)及應(yīng)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(jìn)行初始磷酸化。因此UL97基因突變可以引起HCMV對GCV發(fā)生耐藥。臨床絕大多數(shù)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(fā)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(fā)生突變引起,絕大多數(shù)為發(fā)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(fā)揮抗病毒作用。UL54基因耐藥突變可表現(xiàn)為耐GCV、CDV及FOS[20-26],其突變位于高度保守區(qū),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(xì)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(yǎng)病毒,然后在系列濃度藥物的細(xì)胞培養(yǎng)中檢測病毒的復(fù)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(biāo)法[27]。PRA即病毒在系列濃度藥物的培養(yǎng)基中生長7-10天,然后根據(jù)各藥物濃度下的病毒蝕斑數(shù)來確定藥物的半數(shù)抑制濃度(IC50)。此方法獲得實驗結(jié)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(shù),還可通過利用PCR檢測病毒DNA、免疫組化檢測HCMV pp65抗原表達(dá)、流式細(xì)胞術(shù)檢測病毒感染細(xì)胞[28]以及報告細(xì)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(jìn)行病毒培養(yǎng),從而限制了表型檢測在臨床上的應(yīng)用。表現(xiàn)檢測中的重組表型檢測是確定臨床分離株中發(fā)現(xiàn)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(zhuǎn)移到參考病毒株上,再進(jìn)行耐藥表型檢測。目前技術(shù)發(fā)展到可以利用獨特的限制性內(nèi)切酶位點進(jìn)行靶基因位點特異性重組[8],運用報告基因進(jìn)行快速病毒定量[30]?？寺CMV全基因組的細(xì)菌人工染色體被構(gòu)建為含UL97的變異體,利用此系統(tǒng)也可以進(jìn)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(jìn)行病毒培養(yǎng),存在如下缺點:①耗時費力;②缺乏統(tǒng)一標(biāo)準(zhǔn);③室內(nèi)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(yīng)用有限。

表1 HCMV相關(guān)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(tài)性分析(PCR RFLP)、PCR直接測序法、熒光標(biāo)記雜交雙探針PCR溶解曲線法(FH-PCR-MC)、分子信標(biāo)PCR溶解曲線法和擴(kuò)增阻礙突變系統(tǒng)實時PCR法(ARMS RT PCR)等。PCR RFLP方法是根據(jù)已知HCMV基因組中與藥物抗性有關(guān)DNA序列的突變可丟失或創(chuàng)造一個限制性內(nèi)切酶位點,先進(jìn)行PCR再利用內(nèi)切酶酶切分析[5,12,33]。有時基因突變沒有改變限制性內(nèi)切酶的位點時,可通過引物設(shè)計引入一個限制性內(nèi)切酶位點。PCR RFLP檢測方法優(yōu)點是較快速(2天出結(jié)果)、敏感(突變株達(dá)到10%即可檢測到),且不需培養(yǎng)病毒(可直接檢測標(biāo)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(qū)分。此方法依靠凝膠電泳判斷結(jié)果,敏感性較差,假陰性率較高。由于需要取PCR產(chǎn)物進(jìn)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(jìn)行PCR擴(kuò)增,再對PCR擴(kuò)增產(chǎn)物進(jìn)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(rèn)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(wù)的專業(yè)化和商品化,測序結(jié)果變得準(zhǔn)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(yīng)用價值。但PCR產(chǎn)物直接測序?qū)Ω腥静《局袃?yōu)勢株的檢測效果較好,較難發(fā)現(xiàn)非優(yōu)勢株,對混合株的感染檢出受限,從而限制了其臨床應(yīng)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(shù)和熒光共振能量轉(zhuǎn)移(FRET)原理,結(jié)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(kuò)增產(chǎn)物的溫度,可測定PCR產(chǎn)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(jiān)測PCR后解鏈過程中熒光信號的變化,操作簡便,特異性和準(zhǔn)確性較好,能區(qū)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(jìn)行定量,且容易出現(xiàn)假陽性結(jié)果。分子信標(biāo)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(biāo)替代了雙探針。分子信標(biāo)不像雙探針,由于其莖部不能和模板完全配對結(jié)合,因此穩(wěn)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(biāo)PCR溶解曲線法。除了上述方法,本實驗室建立了ARMS PCR結(jié)合SYBR GreenⅠ的實時PCR法[41]。實時ARMS PCR方法的關(guān)鍵技術(shù)在于引物的設(shè)計。一般檢測單個位點突變需設(shè)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(jié)合突變引物要求能有效擴(kuò)增突變模板,對于野生模板不擴(kuò)增或低效擴(kuò)增;公共引物結(jié)合針對混合模板的引物則可以同時擴(kuò)增突變和野生模板。ARMS PCR引物設(shè)計一般采用引物3'端最后一個堿基錯配可引起產(chǎn)物銳減的原理進(jìn)行設(shè)計。但僅一個堿基錯配往往不能有效阻止野生株的擴(kuò)增,此時需要在引物3'端倒數(shù)第二個堿基上引入額外的錯配堿基才可能達(dá)到有效阻止野生株的擴(kuò)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(biāo)等進(jìn)行實時監(jiān)測。SYBR GreenⅠARMS PCR方法簡單、費用低廉,但其易受非特異性擴(kuò)增產(chǎn)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(biāo)的ARMS實時PCR則排除非特異性擴(kuò)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(biāo)可對病毒突變株占病毒總量的百分比進(jìn)行檢測,并可對突變株進(jìn)行準(zhǔn)確定量[44,45]?？傊?基因型檢測不需病毒培養(yǎng),可直接采用臨床標(biāo)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(shù)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(yán)重的問題。臨床必須不斷了解HCMV耐藥機(jī)制和檢測方法,從而監(jiān)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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