輕或重癥手足口病患兒HEV71分離鑒定及其VP1基因型分析

蔣卓婧，何婷婷，陳金堃，嚴(yán)　杰

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輕或重癥手足口病患兒HEV71分離鑒定及其VP1基因型分析

蔣卓婧1,2，何婷婷2，陳金堃2，嚴(yán)杰1

1. 浙江大學(xué)醫(yī)學(xué)院病原生物學(xué)系，杭州310058；2. 浙江省紹興市疾病預(yù)防控制中心，紹興312071

摘要：目的從不同病情手足口病(HFMD)患兒中分離鑒定人腸道病毒71型(HEV71)并分析其VP1基因型及藥物結(jié)合位點(diǎn)突變情況。方法從2014年浙江省紹興地區(qū)輕、重癥各4例HFMD患兒標(biāo)本中分離HEV71并用RT-PCR進(jìn)行鑒定。采用RT-PCR擴(kuò)增病毒分離株全長(zhǎng)VP1基因并測(cè)序。采用專業(yè)生物信息學(xué)軟件，對(duì)HEV71分離株進(jìn)行VP1基因分型、確定VP1基因中藥物結(jié)合位點(diǎn)突變情況及構(gòu)建HEV71分離株系統(tǒng)進(jìn)化樹(shù)。結(jié)果8例HFMD患兒標(biāo)本中均分離出HEV71。HEV71分離株與文獻(xiàn)報(bào)道的VP1-C4a基因亞型的核苷酸和氨基酸序列相似性分別高達(dá)97.2%～98.8%和99.3%～99.7%。2例重癥患兒HEV71分離株分別在VP1基因序列第179位藥物結(jié)合位點(diǎn)發(fā)生V179L點(diǎn)突變。5株HEV71與安徽和湖南分離株、3株與上海和山東分離株親緣關(guān)系較近。結(jié)論本地區(qū)不同病情HFMD患兒分離的HEV71均為VP1-C4a基因亞型，重癥患兒HEV71分離株VP1基因第179位藥物結(jié)合位點(diǎn)突變可能與病情嚴(yán)重程度有關(guān)。

關(guān)鍵詞：手足口??；人腸道病毒71型；VP1基因；基因分型；藥物結(jié)合位點(diǎn)/突變

人腸道病毒71型(humanenterovirus71，HEV71)為腸道病毒A組單股正鏈RNA病毒，是手足口病(hand-foot-and-mouthdisease，HFMD)主要病原體[1-3]。HEV71殼粒由VP1～VP4 4種病毒蛋白組成，其中VP1具有細(xì)胞受體結(jié)合位點(diǎn)、中和抗體表位及抗病毒藥物結(jié)合位點(diǎn)，故在病毒吸附、穿入、脫殼等感染過(guò)程以及抗病毒免疫應(yīng)答、藥物治療效果等方面發(fā)揮重要作用[4-6]。近年文獻(xiàn)報(bào)道，若VP1功能相關(guān)重要位點(diǎn)發(fā)生突變，可使HEV71毒力和耐藥性發(fā)生改變，從而對(duì)HFMD患者病情嚴(yán)重程度產(chǎn)生明顯影響[7-9]。業(yè)已發(fā)現(xiàn)，HEV71的VP1序列中有一個(gè)微小RNA病毒衣殼蛋白樣功能結(jié)構(gòu)域(picornavirus capsid protein domain-like，rhv_like)，該結(jié)構(gòu)域含有9個(gè)藥物結(jié)合位點(diǎn)[10-12]。此外，根據(jù)VP1序列差異可將HEV71分為A、B0～5和C1～5基因型，不同地區(qū)流行的優(yōu)勢(shì)VP1基因型明顯不同[13-15]。本研究中，我們從當(dāng)?shù)剌p癥或重癥HFMD患兒標(biāo)本中分離并鑒定了HEV71株，繼而測(cè)定并分析了HEV71分離株的VP1基因序列，以期了解本地區(qū)優(yōu)勢(shì)流行的HEV71基因型以及VP1藥物結(jié)合位點(diǎn)突變與患兒病情嚴(yán)重程度之間的關(guān)系。

1材料與方法

1.1病例來(lái)源、診斷及分類2014年紹興地區(qū)未經(jīng)治療的HFMD初診患者。參照我國(guó)衛(wèi)生部2013年版《手足口病診療指南》[16]，根據(jù)患兒臨床表現(xiàn)及實(shí)時(shí)熒光RT-PCR檢測(cè)結(jié)果等進(jìn)行疾病診斷。輕癥病例判斷標(biāo)準(zhǔn)：手、足、口、臀部皮疹可伴發(fā)熱；重癥病例判斷標(biāo)準(zhǔn)：有神經(jīng)系統(tǒng)受累臨床表現(xiàn)或出現(xiàn)下列情況之一者：1)頻繁抽搐、昏迷、腦疝；2)呼吸困難、紫紺、血性泡沫痰、肺部羅音等；3)休克等循環(huán)功能不全表現(xiàn)。4例輕癥或4例重癥HFMD患兒中，均為男性3例、女性1例，年齡1～3歲，前者編號(hào)為SXQ1～4，后者編號(hào)為SXZ1～4[16]。

1.2RT-PCR根據(jù)我國(guó)衛(wèi)生部2013年版《手足口病診療指南》[16]，采集上述HFMD患兒鼻咽拭子、肛拭子和/或皰疹液標(biāo)本。根據(jù)High Pure Viral Nucleic Acid Kit(Roche)說(shuō)明書(shū)，提取上述標(biāo)本中的總RNA。根據(jù)One Step PrimerScript RT-PCR Kit(TaKaRa)說(shuō)明書(shū)，以總RNA為模板，先逆轉(zhuǎn)錄生成cDNA，然后分別用人腸道病毒(HEV)和HEV71引物及其探針，對(duì)逆轉(zhuǎn)錄產(chǎn)物進(jìn)行擴(kuò)增及檢測(cè)。反應(yīng)總體積25 μL，反應(yīng)程序：42 ℃ 30 min，95 ℃ 2 min；95 ℃ 5 s、55 ℃ 35 s，40個(gè)循環(huán)，被檢樣本Ct值≤35.0判為陽(yáng)性。HEV 5′-非編碼區(qū)通用PCR引物和探針序列：上游引物5′-CTG YRG CGG AAC CGA CTA C-3′(Y=C或T，R=G或A)，下游引物5′-ATT GTC ACC ATA AGC AGC CA-3′，探針5′-FAM-TTG GGT GTC CGT GTT T-MGB-3′[17]。HEV71-VP1基因PCR引物和探針序列：上游引物5′-TGA TTG AGA CAC GST GTG TYC TTA-3′(S=G或C)，下游引物5′-CCC GCT CTG CTG AAG AAA CT-3′，探針5′-FAM-TCG CAC AGC ACA GCT GAG ACC ACT C-MGB-3′[18]。

1.3病毒分離與鑒定根據(jù)我國(guó)衛(wèi)生部2012年版《手足口病預(yù)防控制指南》[19]，采集上述8例HFMD患兒鼻咽拭子、肛拭子和/或皰疹液標(biāo)本置于保存液(MEM 80.5 mL，3%L-谷氨酰胺1.0 mL，胎牛血清5 mL，7.5%NaHCO3溶液3.5 mL，10 000 U/mL青霉素和鏈霉素各10 mL)中。非洲綠猴腎細(xì)胞(Vero)株由浙江省疾病預(yù)防控制中心提供，采用含10%小牛血清、100 U/mL青霉素、100 μg/mL鏈霉素的MEM培養(yǎng)液(Gibco)培養(yǎng)。Vero細(xì)胞接種于6孔細(xì)胞培養(yǎng)板中，37 ℃、5%CO2條件下預(yù)培養(yǎng)24 h，使之形成單層細(xì)胞，然后接種病毒保存液，繼續(xù)培養(yǎng)5 d，每天用倒置光學(xué)顯微鏡觀察，若出現(xiàn)細(xì)胞病變(cytopathic effect，CPE)者判為HEV71陽(yáng)性，CPE陰性培養(yǎng)物盲傳兩代仍未出現(xiàn)CPE者判為陰性。按上法提取CPE陽(yáng)性培養(yǎng)物總RNA并用RT-PCR檢測(cè)其病毒及其類型[17-19]。1.4全長(zhǎng)HEV71-VP1基因擴(kuò)增按上法提取8例HFMD患兒HEV71分離株總RNA。根據(jù)Titan One Tube RT-PCR試劑盒(Roche)說(shuō)明書(shū)，先以O(shè)ligo-dT為引物進(jìn)行逆轉(zhuǎn)錄，然后以逆轉(zhuǎn)錄產(chǎn)物為模板，擴(kuò)增全長(zhǎng)HEV71-VP1基因的DNA片段。上游引物：5′-GCA GCC CAA AAG AAC TTC AC-3′；下游引物：5′-AAG TCG CCA GAG CTG TCT TC-3′[14]。反應(yīng)總體積50 μL，反應(yīng)程序：95 ℃ 3 min；95 ℃ 15 s、58 ℃ 15 s、72 ℃ 1.5 min，30個(gè)循環(huán)；72 ℃ 10 min。擴(kuò)增產(chǎn)物委托杭州華大基因公司測(cè)序。

1.5HEV71-VP1基因序列分析參照GenBank中HEV71參考標(biāo)準(zhǔn)株VP1基因A、B0～5、C1～5基因型序列，根據(jù)HEV71分離株全長(zhǎng)VP1基因測(cè)序結(jié)果，采用Mega5.2軟件確定其VP1基因型并構(gòu)建系統(tǒng)進(jìn)化樹(shù)。采用病毒蛋白結(jié)構(gòu)分析軟件(http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)，確定HEV71-VP1中藥物結(jié)合位點(diǎn)及HEV71分離株VP1基因序列中相關(guān)位點(diǎn)突變情況。

2結(jié)果

2.1HFMD患兒臨床診斷SXQ1～4 4例輕癥HFMD患兒有典型皮疹并伴發(fā)熱，SXZ1～4 4例重癥HFMD患兒尚有神經(jīng)或呼吸系統(tǒng)癥狀和體征，但所有患兒標(biāo)本HEV和HEV71 RT-PCR檢測(cè)結(jié)果均為陽(yáng)性(圖1)，表明上述患兒均感染了HEV71。

圖1　8例HFMD患兒標(biāo)本中HEV和HEV71 RT-PCR檢測(cè)結(jié)果Fig.1　RT-PCR detection results of HEV and HEV71 in samples of eight HFMD children

2.2HFMD患兒病毒分離與鑒定結(jié)果SXQ1～4 4例輕癥及SXZ1～4 4例重癥HFMD患兒標(biāo)本中分

離的病毒株RT-PCR檢測(cè)結(jié)果顯示均為HEV71，與患兒臨床標(biāo)本RT-PCR直接檢測(cè)結(jié)果相符。上述HFMD患兒標(biāo)本中HEV71感染Vero細(xì)胞3 d后，出現(xiàn)典型的細(xì)胞圓縮、分散、折光性增強(qiáng)甚至脫落等CPE現(xiàn)象(圖2)。

2.3HEV71分離株VP1基因型分析結(jié)果與GenBank中HEV71各基因型參考標(biāo)準(zhǔn)株VP1基因序列(基因型/登錄號(hào)：A/U22521、B0/AB524081、B1/AB524096、B2/AF135888、B3/AF376074、B4/AF376111、B5/DQ341364、C1/AB524215、C2/JQ621841、C3/JN874555、C4a/EU753409、C4b/AF302996、C5/AM490163)比較，SXQ1～4四例輕癥及SXZ1～4四例重癥HFMD患兒標(biāo)本中分離的8株HEV71株VP1基因核苷酸及氨基酸序列均與C4a基因型相似性最高，分別為97.2%～98.8%和99.3%～99.7%(表1)，表明所有患兒均感染了VP1-C4a基因型HEV71。

表1HEV71分離株VP1基因型比較結(jié)果

Tab.1Comparison results of VP1 genotypes of HEV71 isolates

基因型genotype核苷酸/氨基酸序列相似性(%)theidentitiesofnucleotideandaminoacidsequences(%)SXQ-1SXQ-2SXQ-3SXQ-4SXZ-1SXZ-2SXZ-3SXZ-4A81.9/94.381.4/94.682.4/95.382.3/94.982.2/94.382.0/94.682.0/94.381.7/94.3B084.6/96.384.9/96.985.2/96.684.7/96.384.7/96.384.3/95.984.9/96.384.3/96.9B184.4/96.683.9/97.384.9/96.984.9/96.684.7/96.683.8/96.384.9/96.684.3/97.3B284.1/96.383.8/96.984.6/96.684.6/96.384.2/96.383.8/95.984.6/96.384.2/96.9B384.5/96.683.8/96.984.6/96.984.2/96.684.6/96.684.5/96.384.9/96.684.2/96.6B483.5/96.983.5/97.683.4/97.383.6/96.983.4/96.983.4/96.683.9/96.983.5/97.3B583.6/96.983.2/96.983.7/97.383.5/96.983.7/96.983.6/96.683.8/96.983.2/96.6C189.8/98.989.1/98.990.1/98.789.2/98.990.0/98.989.2/98.789.6/98.988.9/98.7C288.3/98.387.9/98.488.2/98.388.2/98.788.8/98.387.8/97.988.6/98.387.9/97.9C388.8/98.388.7/98.989.1/97.988.4/98.388.8/98.387.9/97.988.8/98.388.3/98.7C4a98.7/99.797.3/99.798.8/99.398.1/99.798.4/99.797.4/99.397.2/99.797.4/99.3C4b92.7/96.992.0/96.992.7/96.692.0/96.993.2/96.992.4/96.692.0/96.992.3/97.3C587.9/98.787.9/98.787.8/98.387.9/98.788.1/98.787.3/98.388.2/98.986.9/98.3

2.4HEV71分離株VP1基因突變?cè)贖EV71-VP1基因rhv_like功能結(jié)構(gòu)域中，有I113、A133、F135、F137、F155、V179、Y201、M230和M253 9個(gè)藥物結(jié)合位點(diǎn)(圖3)。盡管HEV71分離株VP1基因序列較為保守，但2株分離自重癥HFMD患兒病毒株VP1基因第179位藥物結(jié)合位點(diǎn)由纈氨酸(V)突變?yōu)榱涟彼?L)，即出現(xiàn)V179L突變，其余2株分離自重癥HFMD患兒以及所有4株分離自輕癥HFMD患兒HEV71株VP1基因中藥物結(jié)合位點(diǎn)均未發(fā)生突變(圖4)。

圖3　HEV71-VP1基因中功能結(jié)構(gòu)域及藥物結(jié)合位點(diǎn)Fig.3　Domains and drug-binding sites in VP1 genes of HEV71

注：(2)～(5)分別為輕癥患兒HEV71分離株SXQ1～4，(6)～(9)分別為重癥患兒HEV71分離株SXZ1～4；黑底白色者為9個(gè)藥物結(jié)合位點(diǎn)圖4　8株HEV71分離株VP1基因氨基酸序列比較Fig.4　Amino acid sequence comparison of VP1 genes from eight HEV71 isolates

2.5HEV71分離株VP1基因系統(tǒng)進(jìn)化樹(shù)分析結(jié)果與國(guó)內(nèi)外不同地區(qū)HEV71分離株VP1基因比較，3株分離自重癥HFMD患兒標(biāo)本及2株分離自輕癥HFMD患兒標(biāo)本的HEV71株(SXZ2～4，SXQ1和SXQ2)與安徽、湖南地區(qū)HEV71分離株親緣關(guān)系較近，1株分離自重癥HFMD患兒標(biāo)本及2株分離自輕癥HFMD患兒標(biāo)本的HEV71株(SXZ1，SXQ3和SXQ4)與上海、山東地區(qū)HEV71分離株親緣關(guān)系較近(圖5)。

圖5　不同地區(qū)HEV71分離株VP1基因系統(tǒng)進(jìn)化樹(shù)分析Fig.5　Analysis about phylogenetic evolution tree of VP1 genes in HEV71 isolates from different areas

3討論

HFMD是由多種腸道病毒感染引起的兒童常見(jiàn)新發(fā)傳染病(emerging infectious disease)，其病原體有HEV71、柯薩奇病毒(coxasckievirus，CV)A組(CVA 2、4、5、6、10、16型)和B組(CVB 1～5型)、?？刹《?echovirus，ECHO)等，但以HEV71和CVA16較為常見(jiàn)[2,20]。多數(shù)HFMD患兒臨床癥狀和體征輕微，以手、足、口腔等處皮疹或皰疹及發(fā)熱為主要特征，少數(shù)重癥患兒可因并發(fā)神經(jīng)系統(tǒng)病變、呼吸道感染、腦炎、心肌炎等導(dǎo)致死亡[16]。VP1不僅是HEV71主要衣殼蛋白, 也是腸道病毒血清型分型依據(jù)[9]。根據(jù)VP1基因核苷酸序列差異，HEV71分為A(BrCr原型株)、B0～5和C1～5基因型，其中C4基因型又可分為C4a和C4b亞型[21-25]，不同地區(qū)優(yōu)勢(shì)流行的VP1基因型明顯不同[13-15]。歐美國(guó)家主要流行C1和C2基因型，東南亞國(guó)家以B3～B5基因型為主，香港、臺(tái)灣地區(qū)和韓國(guó)以C4和C5基因型為主[15,20-23]。我國(guó)1998年以來(lái)主要流行的HEV71一直是C4基因型, 但2004年前以C4b亞型為主，此后以C4a為主[14, 24]。本研究中來(lái)自4例輕癥和4例重癥HFMD患兒的HEV71分離株VP1基因序列分析結(jié)果顯示，所有HEV71分離株VP1基因核苷酸及氨基酸序列與C4a基因型相似性最高(97.2%～98.8%和99.3%～99.7%)，表明本地區(qū)主要流行的HEV71仍然是C4a基因型，且基因型與患兒病情嚴(yán)重程度無(wú)明顯相關(guān)性。

HFMD患兒病情嚴(yán)重程度，不僅與其遺傳因素、抗感染免疫力、病毒感染量及早期就診等有關(guān)，HEV71抗藥性以及不同HEV71株毒力強(qiáng)弱也可對(duì)患兒病情產(chǎn)生較大影響[16,25]。HEV71-VP1基因rhv_like功能結(jié)構(gòu)域中有9個(gè)藥物結(jié)合位點(diǎn)，若發(fā)生突變，可影響抗病毒藥物治療效果，從而對(duì)HFMD患兒病情產(chǎn)生不良影響[10-12]。我們的實(shí)驗(yàn)結(jié)果顯示，2株分離自重癥患兒HEV71株(SXZ1和SXZ2) VP1基因中第179位藥物結(jié)合位點(diǎn)出現(xiàn)V179L突變，可能與病情嚴(yán)重程度有關(guān)。此外，SXQ2和SXZ4分離株VP1基因E145Q突變、SXZ2分離株VP1基因Q172L突變雖不在藥物結(jié)合位點(diǎn)，但在藥物結(jié)合位點(diǎn)區(qū)域內(nèi)，是否與病毒抗藥性有關(guān)有待于進(jìn)一步研究。

系統(tǒng)進(jìn)化樹(shù)反映病原微生物分離株之間的親緣關(guān)系，可在病原微生物傳染源溯源中發(fā)揮重要作用。我們的VP1基因系統(tǒng)進(jìn)化樹(shù)分析結(jié)果顯示，3例重癥患兒標(biāo)本中分離的HEV71株SXZ2～4、2例輕癥患兒標(biāo)本中分離的HEV71株SXQ1和SXQ2與安徽、湖南地區(qū)分離的HEV71株親緣關(guān)系較近，但1例重癥患兒標(biāo)本中分離的HEV71株SXZ1、2例輕癥HFMD患兒標(biāo)本中分離的HEV71株SXQ3和SXQ4與上海、山東地區(qū)分離的HEV71株親緣關(guān)系較近，提示本地區(qū)流行的HEV71來(lái)源復(fù)雜，這可能與本地區(qū)經(jīng)濟(jì)發(fā)達(dá)及人群流動(dòng)性大有關(guān)。

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DOI:10.3969/j.issn.1002-2694.2016.05.003

通訊作者：嚴(yán)杰，Email: med_bp@zju.edu.cn

中圖分類號(hào)：R373

文獻(xiàn)標(biāo)識(shí)碼：A

文章編號(hào)：1002-2694(2016)05-429-06

收稿日期：2015-12-22修回日期：2016-02-13

Isolation and identification of HEV71 strains from children with hand-foot-and-mouth disease in different conditions and analysis of their VP1 genotypes

JIANG Zhuo-jing1,2, HE Ting-ting2, CHEN Jin-kun2, YAN Jie1

(1.DepartmentofMedicalMicrobiologyandParasitology,ZhejiangUniversitySchoolofMedicine,Hangzhou310058,China;2.ShaoxingCenterforDiseaseControlandPrevention,Shaoxing312071,China)

Abstract:The aim of this study was to isolate the human enterovirus 71 (HEV71) strains from the hand-foot-and-mouth disease (HFMD)-suffering children with different pathogenetic conditions, and the VP1 genotypes of the viral isolates and the mutation of drug-binding sites in the VP1 genes were amalyzed. Firstly, HEV71 strains were isolated from the samples of four mild and four severe HFMD children, and then the viral isolates were identified by RT-PCR. The entire VP1 genes in the isolated HEV71 strains were amplified by RT-PCR for sequencing. Using professional bioinformatic softwares, the VP1 genotypes of the HEV71 isolates and the mutation of drug-binding sites in the VP1 genes were determined, and then the VP1 gene-based phylogenetic evolution tree of these viral isolates was generated. The results showed that HEV71 strains could be isolated from all the samples of the eight cases. Compared with the reported VP1-C4a genotype of HEV71, the identities of nucleotide and amino acid sequences of the viral isolates were 97.2%-98.8% and 99.3%-99.7%, respectively. The mutation at 179th drug-binding site in the VP1 genes (V179L) from two of the four severe cases was found. Five or three of the eight isolates presented the higher genetic relationship with those from Anhui and Hunan, or Shanghai and Shandong areas. All the results led to the conclusions that all the HEV71 strains isolated from the HFMD children in the local area with different pathogenetic conditions belong to VP1-C4a genotype, and the mutation at 179th drug-binding site may be associated with severity of the disease.

Key words:hand-foot-mouth disease/ HFMD; human enterovirus 71/ HEV71; VP1 gene; genotyping; drug-binding site/ mutation Corresponding author: Yan Jie, Email: med_bp@zju.edu.cn