阿米巴巨大病毒及其潛在致病性研究進(jìn)展

夏宇程，鐘江

復(fù)旦大學(xué)生命科學(xué)學(xué)院微生物學(xué)與微生物工程系,上海 200438

阿米巴是自然界中廣泛分布的單細(xì)胞真核生物，通過偽足來捕捉和吞噬顆粒狀物質(zhì)作為食物，被稱為自然界的“巨噬細(xì)胞”。許多人類病原體，如引起軍團(tuán)病的嗜肺軍團(tuán)菌，可在阿米巴細(xì)胞內(nèi)繁殖。因此，阿米巴也有“人類病原體訓(xùn)練場”的稱號[1]。

1 阿米巴巨大病毒的發(fā)現(xiàn)

1996年，有研究者采用與阿米巴共培養(yǎng)技術(shù)，從肺炎暴發(fā)地水樣中分離了多種“軍團(tuán)菌樣阿米巴內(nèi)寄生菌”[2]，其中一種革蘭染色陽性球菌——“Bradford球菌”引起特別關(guān)注[3]，因?yàn)檠芯空邆兪冀K無法從中擴(kuò)增出16S rDNA。直到2003年，La Scola 等通過電子顯微鏡觀察、全基因組測序及阿米巴細(xì)胞裂解周期研究發(fā)現(xiàn)，“Bradford球菌”并非細(xì)菌，而是一種病毒，將其命名為Mimivirus[4]，意為“模擬細(xì)菌的病毒”， 中文譯為擬菌病毒。這種病毒呈二十面體狀，直徑達(dá)400 nm，是當(dāng)時(shí)報(bào)道的最大病毒，染色后可直接在光學(xué)顯微鏡下觀察到。其基因組同樣巨大，為長達(dá)1.18 Mb 的雙鏈DNA，預(yù)測編碼1 262個(gè)開放讀碼框。

此后，人們采用阿米巴共培養(yǎng)方法，從世界各地的淡水、海水、土壤、生物(昆蟲、環(huán)節(jié)動(dòng)物、人體等)樣本中分離到馬賽病毒(Marseillevirus)、Megavirus、潘多拉病毒(Pandoravirus)等多種巨大病毒[5-6]。這些病毒均具有巨大的病毒顆粒(0.3～1.5 μm)和龐大的基因組(最大的潘多拉病毒基因組達(dá)2.5 Mb，最小的馬賽病毒也有0.37 Mb)。而且，一些環(huán)境微生物宏基因組研究結(jié)果證明，這些巨大病毒與其宿主阿米巴一樣分布廣泛[7]。

分析這些巨大病毒的基因組，發(fā)現(xiàn)它們均編碼了其他病毒中罕見的與蛋白質(zhì)合成相關(guān)的基因，包括tRNA、氨酰tRNA合成酶、轉(zhuǎn)譯因子等[8]。最新發(fā)現(xiàn)的一種巨大病毒Klosneuvirus，甚至帶有全部20種氨基酸的特異性氨酰tRNA合成酶[9]。這激發(fā)了演化生物學(xué)家的廣泛興趣，也在學(xué)術(shù)界引起了一場關(guān)于病毒起源和本質(zhì)的大討論[8](表1)。

由于巨大病毒最早發(fā)現(xiàn)于肺炎流行地，許多研究者致力于研究此類病毒對人體的潛在感染和致病性。本文主要介紹這方面的研究進(jìn)展。

表1阿米巴巨大病毒代表株
Tab.1Typicalstrainsofgiantvirusesinamoebae

YearsofsamplecollectionVirusSampletypeCharacteristicsofvirusparticleCharacteristicsofgenomeSize(Mb)CG%ORFSensitivehostsRef．2003MimivirusWaterfroma coolingtowerIcosahedral?like，～400nmindiameter1．1828%1262A．polyphaga[4]2009MarseillevirusWaterfrom acoolingtowerIcosahedral?like，～250nmindiameter0．3745%457A．polyphaga[10]2011MegavirusCoastalwaterIcosahedral?like，～680nmindiameter1．2625%1120A．castellanii[11]2013PandoravirusCoastalsedimentsOvoid，1μminlength，0．5μmindiameter1．91?2．4725%1120A．castellanii[12]2014PithovirusPermafrostsoilOvoid，1．5μminlength，0．5μmindiameter0．636%467A．castellanii[13]2015FaustovirusSewageIcosahedral?like，～500nmindiameter0．4636%451Vermamoeba vermiformis[14]2017KlosneuvirusSludgeIcosahedral?like，～300nmindiameter1．5728%355Notisolated[9]

2 人體感染阿米巴巨大病毒的血清學(xué)和分子診斷證據(jù)

表2匯總了在人體樣本中用血清學(xué)和分子生物學(xué)方法進(jìn)行阿米巴巨大病毒檢測的結(jié)果。大多數(shù)研究在相當(dāng)比例的樣本中檢測到了阿米巴巨大病毒特異性抗體或基因序列，但也有研究未發(fā)現(xiàn)該類病毒存在的證據(jù)。

2.1 擬菌病毒

La Scola等檢測了376例醫(yī)源性肺炎患者的血液樣本，發(fā)現(xiàn)9.7%的樣本中有抗擬菌病毒抗體，相比之下，511例健康人的血液樣本中僅2.3%呈陽性[15]，且抗擬菌病毒抗體水平高于抗“阿米巴相關(guān)細(xì)菌”的抗體[35]；26例重癥監(jiān)護(hù)室(intensive care unit，ICU)源性肺炎患者血液樣本中的抗體陽性率達(dá) 19.2%，從1例患者的支氣管肺泡灌洗液中還檢測到病毒DNA片段，而50例對照者的樣本全為陰性。Berger等采用熒光定量聚合酶鏈反應(yīng)(polymerase chain reaction，PCR)檢測了157例醫(yī)院/社區(qū)獲得性肺炎患者的210份支氣管肺泡灌洗液樣品，發(fā)現(xiàn)19%含有與阿米巴相關(guān)的微生物，擬菌病毒陽性率為7.1%，居肺炎病因的第4位[16]。Raoult等報(bào)道了1例在法國馬賽研究擬菌病毒的實(shí)驗(yàn)室工作人員突然患上不明原因的病毒性肺炎，其血液中抗多種擬菌病毒抗體呈陽性，且抗體水平隨病情好轉(zhuǎn)而降低[17]。Bousbia等對97例ICU患者的173份血液樣品進(jìn)行血清學(xué)研究，結(jié)果顯示抗阿米巴相關(guān)微生物的IgG、IgM隨ICU停留時(shí)間延長而增加，其中抗擬菌病毒抗體占10%[18]；Vincent等也報(bào)道了相同現(xiàn)象[19]。

表2阿米巴巨大病毒的血清學(xué)及分子生物學(xué)檢測結(jié)果
Tab.2SummaryoftheserologicalandmolecularbiologicaldataonMimivirusandMarseillevirus

YearsofsamplecollectionCountryPopula?tionsizeMaincharacteristicsofobjectsSampletypePCRAntibodyRef．1985?19972003?2004CanadaFrance88776Adults：CAPAdults：healthyAdults：ICU?acquiredpneumoniaAdults：healthyBloodBloodBALBALN．t．N．t．3．1%09．7%2．3%19．2%0[15]2002?2003France157Adults：ICUpneumoniaBAL7．1%N．t．[16]2004France1Adults：viralpneumoniaBloodN．t．positive[17]2007?2008France97Adults：ICUpneumoniaAdults：admissiontoICUwithoutpneumoniaBloodN．t．IgG：3%IgM/IgG：10%[18]2006?2008France300Adults：VAPBALN．t．19．7%[19]2005?2006Austria214Children：RTINPaspirateA．N．N．t．[20]2003?20042003Australia396Adults/children：ARTIAdults/children：HRVincidenceNPaspirateA．N．N．t．[21]2000?20012003?20042003200220042004200120032002?2003AmericaThailandAmericaCanada496Children：CAPAdults/children：CAPGeriatric：NosocomiallyacquiredpneumoniaAdults/children：CAPAdults：bonemarrowtransplantrecipientsAdults：lungtransplantrecipientsNasalswabsNPswabsNasalswabsBAL，sputum，ETaspirateNPaspirate，NPswabs，NasalswabsNPswabsA．N．N．t．[22]2009?2010Netherland109Adults：COPDSputum，bloodA．N．2．5%[23]2005?2009Netherland214Adults：VAPBALA．N．N．t．[24]2012Italy63Patients：ventilatedfor48hPatients：nonventilatedBALA．N．N．t．[25]2011?20152013?2015China3696Children：ARTIAdults/children：ARTINPaspirateThroatswabs1positiveN．t．N．t．[26]2013France30Adults：healthyAdults：healthyBloodForhigh?throughputseq10%IgG：15%[27]

(續(xù)表2)

N.t.: not told in refence; A.N.: all negative; CAP: community-acquired pneumonia; ICU: intensive care unit; BAL: bronchoalveolar lavage; VAP: ventilator-associated pneumonia; RTI: respiratory tract infection; NP: Nasopharyngeal; ARTI: acute respiratory tract infection; HRV: human rhinovirus; ET: endotracheal; COPD: chronic obstructive pulmonary disease; CSF: cerebrospinal fluid.

醫(yī)院環(huán)境中也檢測到擬菌病毒的存在。dos Santos Silva等對巴西一家大醫(yī)院中多個(gè)場所收集的242份環(huán)境擦拭子樣品進(jìn)行定量PCR檢測[36]，發(fā)現(xiàn)擬菌病毒陽性率為12.4%，其中取自呼吸道疾病隔離設(shè)施樣品的陽性率達(dá)36.4%，且通過阿米巴培養(yǎng)從20%樣品中分離到了病毒。以上這些研究結(jié)果提示，擬菌病毒有可能與人類肺炎等呼吸道疾病有關(guān)。

也有研究結(jié)果不支持?jǐn)M菌病毒與呼吸系統(tǒng)疾病的關(guān)系。Larcher 等[20]、Arden等[21]及Zhang等[26]用多種病毒特異性引物對呼吸道感染患者的鼻咽吸出物/拭子進(jìn)行PCR檢測，檢出副流感病毒、腺病毒等常見呼吸道病毒，但未檢測到擬菌病毒。Dare等[22]、Vanspauwen等[23-24]及Costa等[25]對肺炎患者的支氣管肺泡灌洗液進(jìn)行PCR檢測，也未檢出擬菌病毒的核酸。

2.2 馬賽病毒

馬賽病毒是2009年報(bào)道的采用類似分離擬菌病毒的方式從冷卻水塔內(nèi)分離獲得的另一種巨大病毒[11]。Popgeorgiev等對10份健康獻(xiàn)血者的血液樣品采用高通量測序進(jìn)行病毒組(virome)分析，發(fā)現(xiàn)2.5%讀出序列(reads)與馬賽病毒的基因組相匹配。他們對另20份血樣進(jìn)行血清學(xué)和PCR檢測，發(fā)現(xiàn)其中3份IgG陽性，2份PCR陽性，并從1份IgG和PCR均陽性的血液樣本淋巴細(xì)胞中分離獲得一株新病毒，命名為GBM(giant blood Marseillevirus)[27]。該作者后來進(jìn)一步擴(kuò)大樣本容量，發(fā)現(xiàn)174份健康獻(xiàn)血者樣品中IgG陽性率為12.6%，PCR陽性率為4%；而22例地中海貧血患者中IgG陽性率為22.7%，PCR陽性率為9.1%。作者推測頻繁輸血治療會(huì)提高感染GBM的概率[28]。他們還在1例病因不明單側(cè)腋窩淋巴結(jié)腫大的幼兒淋巴結(jié)切片中，采用熒光原位雜交(fluorescenceinsituhybridization，F(xiàn)ISH)和免疫組化方法檢測到巨噬細(xì)胞胞質(zhì)中存在馬賽病毒，其血液樣本中也能檢測到相應(yīng)抗體和病毒核酸，但13個(gè)月后檢測結(jié)果轉(zhuǎn)為陰性。因?yàn)樵摶純涸歼^卡介苗感染(BCGitis)，推測該病毒對免疫紊亂者有致病性[29]。Aherfi等在1 例發(fā)熱性腸炎患者血液和咽部均檢測到馬賽病毒DNA，患者服用環(huán)丙沙星72 h后癥狀消退，但4個(gè)月后出現(xiàn)眩暈并伴隨體重減輕，血液和咽喉部仍可檢測到該病毒DNA，推測該病毒的存在對神經(jīng)系統(tǒng)有慢性影響[30]。他們還在1例霍奇金淋巴瘤患者淋巴結(jié)內(nèi)檢測到馬賽病毒[31]。Mueller等對517例健康瑞士人的血液進(jìn)行檢測，發(fā)現(xiàn)9例為抗洛桑病毒(Lausannevirus；馬賽病毒科成員)抗體陽性[32]。

同樣，也有研究未在血液樣本中檢測到馬賽病毒[33-34]。針對假陽性的質(zhì)疑，Sauvage等認(rèn)為部分實(shí)驗(yàn)在其他醫(yī)院完成，不太可能存在樣品污染；并指出，即使是陽性的健康人血液樣品，PCR信號仍非常微弱，只有引發(fā)某些疾病(如前述兒童淋巴結(jié)病[29])時(shí)才會(huì)出現(xiàn)PCR強(qiáng)陽性，且患者病愈后檢測不到病毒核酸，但抗體檢測仍為陽性[33]。

3 宏基因組測序檢測阿米巴巨大病毒

隨著高通量測序技術(shù)的成熟和費(fèi)用逐漸降低，許多研究應(yīng)用這項(xiàng)技術(shù)來尋找巨大病毒[7]。馬賽病毒病毒株GBM[27]及馬賽病毒科的另一成員塞內(nèi)加爾病毒(Senegalvirus)[37]就是研究者對人體血液及糞便樣本進(jìn)行高通量測序時(shí)發(fā)現(xiàn)并進(jìn)一步分離獲得的。Verneau等設(shè)計(jì)了MG-Digger軟件，專門從宏基因組數(shù)據(jù)中搜索巨大病毒的序列信息[38]，并分別從Loh等[39]報(bào)道的人血液樣本數(shù)據(jù)庫和Law等[40]報(bào)道的慢性肝炎患者血液樣本數(shù)據(jù)庫中檢測到185和1 706條巨大病毒讀出序列，分別占總讀出序列的0.6%和0.004%。Rampelli等設(shè)計(jì)了搜索人體宏基因組內(nèi)病毒信息的ViromeScan軟件，分析了人體微生物組計(jì)劃(Human Microbiome Project，HMP)中采自不同部位(糞便-腸道、陰道、口腔黏膜、耳后折痕區(qū))的宏基因組庫，均發(fā)現(xiàn)了巨大病毒的序列[41]，且腸道內(nèi)擬菌病毒和痘病毒科占多數(shù)，病毒成分明顯不同于其他部位。其他提及巨大病毒的人體宏基因組研究如表3所示。從中可看出，巨大病毒的信息占比較低，可能是因?yàn)椋翰《窘M研究的樣品制備時(shí)往往采用0.22～0.45 μm濾膜過濾，導(dǎo)致巨大病毒損失[7,45]；巨大病毒基因組內(nèi)有很多未注釋的開放讀碼框，加上病毒基因的多樣性，妨礙了對所建庫中病毒信息的注釋和挖掘[7]。

4 從人體樣本中分離阿米巴巨大病毒

許多研究將樣本與阿米巴進(jìn)行共培養(yǎng)，從而獲得巨大病毒的分離株，詳見表4。常用阿米巴為卡氏棘阿米巴(Acanthamoebacastellanii)、多噬棘阿米巴(Acanthamoebapolyphaga)和蚓狀哈曼阿米巴(Vermamoebavermiformis)[52]。La Scola等從1例角膜炎患者的隱形眼鏡護(hù)理液內(nèi)分離出1株擬菌病毒Lentille[46-47]。Saadi等收集了196例社區(qū)獲得性肺炎患者的不同呼吸道樣本，從1例不明原因肺炎婦女的支氣管抽吸液中分離到1株擬菌病毒LBA111[48]；該作者還收集并檢測了1 605份糞便樣品，從其中1例不明原因肺炎患兒的糞便中分離到1株擬菌病毒Shan virus[49]。Scheid等發(fā)現(xiàn)，從1例角膜炎患者的隱形眼鏡護(hù)理液內(nèi)分離到的棘阿米巴細(xì)胞含有細(xì)菌樣顆粒，會(huì)裂解棘阿米巴的滋養(yǎng)體[50]，PCR和全基因組測序證實(shí)該胞內(nèi)物是1株潘多拉病毒[51,53]。

5 阿米巴巨大病毒感染細(xì)胞及實(shí)驗(yàn)動(dòng)物

研究表明，巨大病毒通過阿米巴細(xì)胞的胞吞作用侵入細(xì)胞[54-55]。哺乳動(dòng)物中，胞吞作用一般由巨噬細(xì)胞、單核細(xì)胞、中性粒細(xì)胞等特定細(xì)胞完成[56]。Ghigo等發(fā)現(xiàn)，擬菌病毒可通過胞吞作用進(jìn)入人髓系細(xì)胞(循環(huán)血單核細(xì)胞、單核細(xì)胞源性巨噬細(xì)胞、單核細(xì)胞系THP-1)和鼠髓系細(xì)胞(骨髓源性巨噬細(xì)胞、巨噬細(xì)胞系RAW264.7、J774A.1)，但無法進(jìn)入非吞噬細(xì)胞(HeLa細(xì)胞等)。擬菌病毒被巨噬細(xì)胞胞吞后，病毒DNA含量隨時(shí)間延長而逐漸增加，并在30 h后引起明顯的細(xì)胞病變，表明巨大病毒不僅可進(jìn)入人巨噬細(xì)胞，還可在其中復(fù)制并引起細(xì)胞病變[55]。Silva等也證實(shí)，擬菌病毒能在人外周血單核細(xì)胞(peripheral blood mononuclear cell，PBMC)中復(fù)制，被感染的細(xì)胞還可產(chǎn)生干擾素，但細(xì)胞受干擾素激活的基因轉(zhuǎn)錄被病毒抑制；如果在病毒感染前預(yù)先用干擾素β1而非干擾素α2處理PBMC，擬菌病毒就無法復(fù)制。這表明干擾素系統(tǒng)能抑制病毒復(fù)制，而擬菌病毒編碼了相應(yīng)的免疫逃避分子以幫助其在宿主細(xì)胞內(nèi)復(fù)制[57]。

表3宏基因組測序分析檢測人體樣本中阿米巴巨大病毒
Tab.3Identificationofsequencescloselyrelatedtogiantvirusesinamoebaeinhumansamplesbymetagenomicanalysis

YearsofsamplecollectionCountryPopulationsizeMaincharacteristicsofobjectsSampletypeMeanRawreadsNumberofhits(%)Ref．2013France30Adults：healthyBloodRoche45420238333(2．5)[28]2012Senegal3Adults：healthyFecesRoche45418285353(0．11)[37]2002?2005Middle East399Adults：AFI/ healthyBloodRoche45429117185(0．6)[38，39]2013Canada11Adults：liver disease/healthyBloodIlluminaGAII427068831706(0．004)[38，40]1978?2005Australia/ America12Children： diarrheaFeces“Micro?mass sequencing”46085(0．001)[42]2011Sweden90Adults：twins， CFSBloodRoche4543051912(0．06)[43]2004?2005Sweden210Adults：RTINP aspirateRoche4547037902(0．0003)[44]

AFI: acute febrile illness; CFS: chronic fatigue syndrome; RTI: respiratory tract infection; NP: nasopharyngeal.

表4人體樣本中分離到的阿米巴巨大病毒株
Tab.4Typicalstrainsofgiantvirusesinamoebaeinhumansamples

YearsofsamplecollectionCountryPopulationsizeMaincharacteristicsofobjectsSampletypeStrainRef．2013France30Adults：healthyBloodMarseillevirus：GBM[28]2011France1Adults：ameba? associatedkeratitisCornealscraping/ contactlensstorageMimivirus：Lentille； Virophage：Sputnik2[46，47]2009?2010Tunisia196Adults：CAPPulmonarysampleMimivirus：LBA111[48]2008?2013France， Tunisia， Senegal1605Children：healthy/ gastroenteritis/CAPStoolMimivirus：Shanvirus[49]2012France， Senegal3Adults：healthy/obeseStoolMarseillevirus： Senegalvirus[37]2008Germany1Adults：keratitisContactlensstoragePandoravirus：KLaHel[50，51]

CAP: community-acquired pneumonia; Pulmonary sample: including bronchial aspirations,bronchoalveolar lavages,lung biopsies,pleural samples.

目前關(guān)于巨大病毒及其組分對動(dòng)物的致病實(shí)驗(yàn)有兩篇報(bào)道。Khan等通過心內(nèi)注射將擬菌病毒接種至小鼠，發(fā)現(xiàn)75%小鼠出現(xiàn)急性肺炎。他們從發(fā)病小鼠的肺部和脾臟分離到擬菌病毒，表明擬菌病毒很可能是肺炎的病原體[58]。Shah等研究了擬菌病毒膠原蛋白L71(一種病毒表面蛋白)在類風(fēng)濕關(guān)節(jié)炎中的潛在作用，用L71免疫膠原性關(guān)節(jié)炎模型小鼠，誘導(dǎo)產(chǎn)生的抗體可與小鼠Ⅱ型膠原蛋白反應(yīng)并引起T細(xì)胞反應(yīng)，導(dǎo)致關(guān)節(jié)炎癥。他們還對100例健康人和100例類風(fēng)濕關(guān)節(jié)炎患者進(jìn)行酶聯(lián)免疫吸附試驗(yàn)(enzyme-linked immunosorbent assay，ELISA)和蛋白免疫檢測，發(fā)現(xiàn)分別有30份和36份血液樣品中病毒核心蛋白L425抗體陽性，同時(shí)分別有6份和22份樣品中L71抗體陽性，據(jù)此認(rèn)為L71是引起類風(fēng)濕關(guān)節(jié)炎的潛在致病因子[59]。

6 結(jié)語

越來越多的研究表明擬菌病毒與肺炎等炎癥有關(guān)，但也有研究不支持這一觀點(diǎn)。陰性結(jié)果的出現(xiàn)不能排除由技術(shù)限制所致：這些研究幾乎只進(jìn)行了PCR，沒有開展血清學(xué)檢測。目前尚缺乏合適的巨大病毒抗原，導(dǎo)致大規(guī)模血清學(xué)篩查難以進(jìn)行。免疫沉淀與高通量測序相結(jié)合的新技術(shù)——VirScan[60]也許可豐富這方面的研究。另一方面，除擬菌病毒和馬賽病毒外，其他阿米巴巨大病毒甚至非阿米巴巨大病毒也有可能對人體造成一定影響[61]。例如，用蚓狀哈曼阿米巴分離獲得的Faustovirus與豬瘟病毒的進(jìn)化地位接近[14]；有研究者在人口咽拭子中檢測到一種分離自綠藻的巨大病毒Acanthocystisturfaceachlorella virus的DNA，且該病毒能在小鼠模型中引起認(rèn)知功能紊亂[62]。此外，目前我國還少見此類病毒的研究?？紤]到不少疾病，如20%～30%的呼吸系統(tǒng)疾病，還未找到致病原因[63]，因此探討巨大病毒等在常規(guī)檢測中易被忽視的微生物的潛在致病性有重要現(xiàn)實(shí)意義。

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