鼠諾如病毒感染免疫研究進(jìn)展

王慧敏 龐立麗 新燕 段招軍

010000 呼和浩特，內(nèi)蒙古醫(yī)科大學(xué)基礎(chǔ)醫(yī)學(xué)院免疫學(xué)(王慧敏、新燕)；102206 北京，中國(guó)疾病預(yù)防控制中心病毒病預(yù)防控制所，衛(wèi)生部醫(yī)學(xué)病毒和病毒病重點(diǎn)實(shí)驗(yàn)室(龐立麗、段招軍)

·綜述·

鼠諾如病毒感染免疫研究進(jìn)展

王慧敏 龐立麗 新燕 段招軍

010000 呼和浩特，內(nèi)蒙古醫(yī)科大學(xué)基礎(chǔ)醫(yī)學(xué)院免疫學(xué)(王慧敏、新燕)；102206 北京，中國(guó)疾病預(yù)防控制中心病毒病預(yù)防控制所，衛(wèi)生部醫(yī)學(xué)病毒和病毒病重點(diǎn)實(shí)驗(yàn)室(龐立麗、段招軍)

王慧敏、龐立麗為并列第一作者

人諾如病毒(Norovirus, NoV)是杯狀病毒家族的成員，可引起以惡心、嘔吐、腹痛和腹瀉為主要臨床癥狀的急性胃腸炎。NoV全球流行，可感染所有年齡階段的人群，造成了嚴(yán)重的疾病負(fù)擔(dān)。由于缺乏合適的小動(dòng)物模型，人類對(duì)NoV的感染免疫和致病機(jī)理還缺乏了解。鼠諾如病毒(Murine norovirus, MNV)最初在免疫缺陷的小鼠中分離得到，在小鼠中可感染和流行，為研究NoV感染引發(fā)的宿主腸道免疫機(jī)制提供了替代模型。本研究從固有免疫和適應(yīng)性免疫兩方面來(lái)闡述。

Fund program: National Natural Science Foundation of China(81601813)

諾如病毒(Norovirus, NoV)原名為諾沃克病毒(Norwalk virus)，由美國(guó)學(xué)者Kapikin在美國(guó)諾瓦克地區(qū)的一所學(xué)校暴發(fā)的胃腸炎患者糞便標(biāo)本中檢出[1]，它是引起人類流行性非細(xì)菌性胃腸炎的主要原因之一。該病毒在全球范圍內(nèi)均有流行，所有年齡階段的人都可以感染，因此造成了巨大的經(jīng)濟(jì)和社會(huì)負(fù)擔(dān)。

NoV具有很強(qiáng)的腸道傳染性，它可以在免疫缺陷患者中持續(xù)感染很長(zhǎng)時(shí)間[2, 3]。到目前為止，NoV傳染性不能被有效阻止。近期研究報(bào)道利用人腸道細(xì)胞培養(yǎng)物成功地培養(yǎng)NoV，但是仍然很難建立體外培養(yǎng)系統(tǒng)并且缺乏動(dòng)物模型，導(dǎo)致NoV感染的發(fā)病機(jī)制和免疫分析受到了阻礙[4]。鼠諾如病毒(Murine norovirus, MNV)的發(fā)現(xiàn)為研究NoV的感染機(jī)制和免疫反應(yīng)提供了模型[5]。

1 鼠諾如病毒的特性

2003年，Karst等[6]在RAG/STAT1-/-免疫缺陷的小鼠中首次分離到MNV。從生物學(xué)和遺傳學(xué)角度方面，MNV與NoV有許多相似性。MNV屬杯狀病毒科NoV屬，是一個(gè)小的無(wú)包膜的正鏈RNA病毒，它屬于NoV基因組的GV。其基因組RNA長(zhǎng)約7.4 kb，有3個(gè)主要的開(kāi)放閱讀框(Open reading frames，ORFs)，分別是ORFl編碼多聚蛋白、ORF2編碼一個(gè)58.9 kDa核衣殼蛋白VPl、ORF3編碼小分子堿性蛋白VP2[7]，2007年，Thackray等通過(guò)對(duì)MNV已知序列的分析，發(fā)現(xiàn)了ORF4，在人、牛、豬NoV中沒(méi)有發(fā)現(xiàn)[8]。2011年，McFadden等發(fā)現(xiàn)ORF4編碼的VF1蛋白參與固有免疫應(yīng)答的調(diào)節(jié)[9]，MNV最主要的毒株有MNV-1(CW1、CW2、CW3、CW4)、MNV-2、MNV-3、MNV-4和MNV-CR6[10]。MNV-CR6和MNV-3MNV毒株可以造成腸道系統(tǒng)持續(xù)感染，這有利于研究病毒持續(xù)感染機(jī)制以及定居在腸道的微生物與宿主免疫系統(tǒng)之間的關(guān)系。Nov和MNV可以與細(xì)胞表面表達(dá)的寡聚糖結(jié)合[11-13]。外周組織的血型抗原稱為組織血型抗原(Histo-blood group antigen, HBGA)，NoV識(shí)別HBGAs，與NoV感染疾病易感性有關(guān)[14, 15]。近期研究發(fā)現(xiàn)CD300lf蛋白和CD300ld蛋白是MNV的受體，MNV不能感染Cd300lf-/-小鼠[16, 17]。

MNV引起的隱性感染在免疫功能正常的小鼠中無(wú)明顯組織病理改變，[6, 18]。MNV感染固有免疫缺陷小鼠后，病理組織檢測(cè)，感染器官有腸、肝、脾、淋巴結(jié)和肺，出現(xiàn)腦炎、肺炎、肝炎等癥狀，產(chǎn)生高致死性[6, 20]。這些數(shù)據(jù)表明，MNV感染與小鼠免疫反應(yīng)關(guān)系密切。

2 鼠諾如病毒免疫學(xué)研究

2.1 鼠諾如病毒的固有免疫 固有免疫對(duì)控制小鼠MNV感染(特別是MNV1)非常重要。特別是干擾素(Interferon, IFN)反應(yīng)對(duì)抑制MNV感染起到重要作用。MNV感染正常小鼠無(wú)臨床癥狀，但是會(huì)導(dǎo)致RAG/STAT1-/-和IFNαβγ-/-小鼠出現(xiàn)高致死性[21]。MNV可以在體外感染巨噬細(xì)胞和樹(shù)突狀細(xì)胞，MNV的增殖受IFN-α和IFN-β受體以及STAT-1限制[18]。啟動(dòng)病毒感染固有免疫應(yīng)答的關(guān)鍵是模式識(shí)別受體(Pattern recognition receptors, PRRs)對(duì)病原體相關(guān)分子模式(Pathogen-associated molecular pattern，PAMP)的識(shí)別。研究表明MNV感染時(shí)，MDA5參與了對(duì)MNV感染的識(shí)別，啟動(dòng)了固有免疫反應(yīng)，MNV-1感染MDA5-/-樹(shù)突狀細(xì)胞時(shí)產(chǎn)生的細(xì)胞因子反應(yīng)是受損的[22]。這種固有免疫信號(hào)導(dǎo)致抗原遞呈細(xì)胞通過(guò)STAT-1通路生產(chǎn)炎性細(xì)胞因子產(chǎn)生了Ⅰ型和Ⅱ型干擾素。在抗MNV免疫應(yīng)答中涉及到細(xì)胞類型和轉(zhuǎn)錄因子，而轉(zhuǎn)錄因子干擾素調(diào)節(jié)因子(Interferon regulatory factor)IRF-3和IRF-7協(xié)同工作啟動(dòng)了獨(dú)特而重疊的抗病毒反應(yīng)來(lái)限制MNV的復(fù)制，IRF-3和IRF-7對(duì)MNV感染巨噬細(xì)胞產(chǎn)生IFN-α/β是必不可少的。Ⅰ型和Ⅱ型干擾素受體以及IRF-3和IRF-7在抗MNV感染固有免疫反應(yīng)是至關(guān)重要的[23]。

固有免疫反應(yīng)抑制病毒復(fù)制，干擾素又是病毒感染的固有免疫反應(yīng)的重要組成部分[24]。干擾素的作用與病毒基因組翻譯的抑制有關(guān)，Ⅰ型和Ⅱ型干擾素抑制巨噬細(xì)胞和樹(shù)突狀細(xì)胞中MNV翻譯。Ⅰ型干擾素,包括IFN -α/β,是由大多數(shù)感染病毒的細(xì)胞產(chǎn)生；Ⅱ型干擾素，IFN-γ是由特定細(xì)胞的免疫系統(tǒng)產(chǎn)生的。Ⅰ型干擾素可能通過(guò)干擾素刺激基因15(ISG15)的活性限制MNV復(fù)制[25]，復(fù)制依賴RNA激活的蛋白激酶[26]。在Ⅰ型干擾素缺失的情況，IFN-γ通過(guò)激活自噬蛋白復(fù)合的Atg5-Atg12/Atg16L1介導(dǎo)的抗病毒活性對(duì)抗MNV，從而抑制巨噬細(xì)胞胞漿內(nèi)的MNV復(fù)制復(fù)合體，而不是通過(guò)自噬相關(guān)途徑進(jìn)行降解[27]。

近期研究表明Ⅲ型干擾素(IFN-λ)與MNV持續(xù)性感染有關(guān)。C57BL/6小鼠感染了MNV-1.CW3后腸系膜淋巴結(jié)和Peyer’s結(jié)細(xì)胞產(chǎn)生IFN-λ[27]。然而，持續(xù)性感染的MNV.CR6不會(huì)誘導(dǎo)Ⅲ型干擾素的生產(chǎn)。外源性IFN-λ治療MNV.CR6持續(xù)感染小鼠，可降低腸道內(nèi)的病毒，最終導(dǎo)致病毒的清除，而無(wú)需啟動(dòng)適應(yīng)性免疫應(yīng)答[28]。研究還發(fā)現(xiàn)小鼠抗生素治療會(huì)增加IFN-λ的抗MNV病毒感染的作用，進(jìn)一步研究表明抗生素治療改變了腸道微生物菌群從而改變了宿主抗MNV病毒固有免疫效應(yīng)[29]。腸道微生物干擾Ⅲ型干擾素，促進(jìn)了MNV的持續(xù)性感染。同時(shí)，腸道RNA病毒可以替代腸道共生菌有益的作用，MNV可以重塑小腸形態(tài)和淋巴細(xì)胞作用[30]。此外，近期研究表明，如果CD11c陽(yáng)性樹(shù)突狀細(xì)胞不能應(yīng)答Ⅰ型干擾素，急性感染病毒株MNV-CW3也會(huì)變成持續(xù)感染[31]。這些研究表明固有免疫不僅僅激活獲得性免疫，固有免疫缺陷也會(huì)造成病毒持續(xù)感染。另外，MNVORF4編碼VF1蛋白，有拮抗固有免疫應(yīng)答的作用。VF1可通過(guò)延遲上調(diào)CXCL10、ISG54、和IFN-β來(lái)增強(qiáng)病毒的復(fù)制[32]。

2.2 鼠諾如病毒的適應(yīng)性免疫應(yīng)答 雖然干擾素和固有免疫反應(yīng)有抑制病毒復(fù)制的作用，但適應(yīng)性免疫是清除病毒的關(guān)鍵。MNV感染B和T細(xì)胞缺陷的RAG1和RAG2基因敲除小鼠，病毒RNA載量升高，病毒持續(xù)感染。B細(xì)胞通過(guò)產(chǎn)生抗體幫助清除病毒。RAG1敲除小鼠注射MNV抗體可以降低病毒滴度[21, 33]。T細(xì)胞免疫對(duì)于清除NoV感染，同產(chǎn)生抗體的B細(xì)胞一樣，也是必須的。在MNV衣殼蛋白免疫小鼠的研究中，抵抗NoV感染需要廣泛地活化T細(xì)胞和B細(xì)胞，包括CD4+和CD8+T細(xì)胞[34]。MNV-1感染小鼠RAW264.7巨噬細(xì)胞，Th1反應(yīng)增加，CCL2、CCL3、CCL4、CCL5、CXCL2、CXCL10以及CXCL11基因表達(dá)均上調(diào)[35]。在體內(nèi)，Ⅰ型和Ⅱ型干擾素誘導(dǎo)產(chǎn)生趨化因子(例如CXCL10和CXCL11)對(duì)運(yùn)輸Th1細(xì)胞很重要[36]。

病毒持續(xù)性感染需要病毒復(fù)制和免疫清除之間的平衡。缺陷的獲得性免疫可以造成病毒持續(xù)感染。特異性CD8+T細(xì)胞應(yīng)答與MNV持續(xù)性感染有關(guān)。病毒特異性CD8+T細(xì)胞活性較低的小鼠感染MNV，無(wú)法清除病毒造成長(zhǎng)期感染，而高水平的CD8+T細(xì)胞活化的小鼠能夠清除MNV感染[37]。CD8+T細(xì)胞是適應(yīng)性免疫應(yīng)答清除MNV感染的另一個(gè)關(guān)鍵組成部分，然而，為了防止MNV再次感染，抗體和CD4+T細(xì)胞更重要。對(duì)MNV-3特異性抗體或CD4+T細(xì)胞的存在足以產(chǎn)生對(duì)MNV-3再次感染的部分保護(hù)，但CD8+T細(xì)胞單獨(dú)并不能免于再次感染MNV-3[38]。

3 結(jié)論

NoV高度變異性，傳播范圍廣，造成的疾病負(fù)擔(dān)嚴(yán)重性，引發(fā)人類高度關(guān)注與重視，但是根據(jù)目前研究進(jìn)展，對(duì)NoV無(wú)有效的治療藥物及疫苗，對(duì)NoV感染免疫特征和致病機(jī)制也不完全清楚。MNV是體外組織培養(yǎng)NoV屬的最佳成員。MNV為NoV分子機(jī)制的研究奠定了基礎(chǔ)，也為NoV免疫機(jī)制、致病機(jī)制的研究和疫苗的制備提供了幫助，對(duì)MNV固有免疫和適應(yīng)性免疫反應(yīng)的探討，進(jìn)一步加深了對(duì)NoV的認(rèn)識(shí)。但是MNV免疫學(xué)機(jī)制還存在許多未知，還需要研究以深入了解NoV與免疫系統(tǒng)的相互關(guān)系。

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Research progress of murine norovirus infection and immunity

WangHuimin,PangLili,XinYan,DuanZhaojun

DepartmentofImmunology,BasicSchoolofMedicalScience,InnerMongoliaMedicalUniversity,Hohhot010110,China(WangHM,XinY);KeyLabortoryofMedicalVirology,MinistryofHealthNationalInstituteforViralDiseaseControlandPrevention,ChineseCentreforDiseaseControlandPrevention,Beijing102206,China(PangLL,DuanZJ)

WangHuiminandPangLiliarethefirstauthorswhocontributedequallytothearticleCorrespondingauthor:XinYan,Email:xinyan_2505@126.com;DuanZhaojun,Email:zhaojund@126.com

Human norovirus (NoV) is a member of the calicivirus family, can cause nausea, vomiting, abdominal pain and diarrhea as the main clinical symptoms of acute gastroenteritis. Human norovirus infection can be popular in the world, all ages, causing serious burden of disease. Due to the lack of suitable small animal models, there is still a lack of understanding of human immune to norovirus infection and pathogenesis. Murine norovirus (MNV) was originally isolated in immune deficient mice, and causes infection and epidemic in mice. MNV provides an alternative model to study human norovirus infection and the host intestinal immune mechanism. This article will elaborate on two aspects of innate immunity and adaptive immunity.

Murine norovirus; Infection; Immune response

新燕，Email：xinyan_2505@126.com；段招軍，Email：zhaojund@126.com

10.3760/cma.j.issn.1003-9279.2017.01.017

鼠諾如病毒；感染；免疫反應(yīng)

國(guó)家自然科學(xué)基金(81601813)

2016-11-29)