本发明涉及病毒领域和肿瘤治疗领域。具体而言，本发明涉及肠道病毒68型(ev-d68)或其修饰形式，或包含ev-d68或其修饰形式的基因组序列或cdna序列，或所述基因组序列或cdna序列的互补序列的核酸分子，用于在受试者(例如，人)中治疗肿瘤的用途，以及在制备用于在受试者(例如，人)中治疗肿瘤的药物中的用途。本发明还涉及一种治疗肿瘤的方法，其包括向有此需要的受试者施用ev-d68或其修饰形式，或包含ev-d68或其修饰形式的基因组序列或cdna序列，或所述基因组序列或cdna序列的互补序列的核酸分子的步骤。
背景技术：
：：目前恶性肿瘤的治疗手段主要包括手术治疗、化疗和放疗。这些传统疗法对于已转移的肿瘤治疗效果不尽如人意，并且还会对患者身体健康产生伤害巨大。相比之下，作为新型的治疗方式，利用溶瘤病毒对肿瘤的治疗方法具有特异性高、效果好、副作用小等特点，是一种目前被认为比较有前景的肿瘤治疗手段。溶瘤病毒是一种能够在肿瘤细胞当中自我复制，从而杀死、溶解肿瘤细胞，或者使肿瘤细胞生长停滞的病毒。当进行体内治疗时，溶瘤病毒表现出对肿瘤细胞的选择特异性，能够直接诱导肿瘤细胞死亡，而对正常细胞影响较小或无影响；同时，溶瘤病毒也能通过激发免疫系统当中的细胞毒性t淋巴细胞反应，间接地杀伤肿瘤细胞。肠道病毒隶属于小rna病毒科，其基因组为单股正链rna。将肠道病毒用作溶瘤病毒有以下几类优点：首先作为单链rna病毒，其基因组在宿主体内没有dna阶段，因此不存在由病毒基因组插入到宿主dna所引起的基因毒性，具有较好的安全性；其次，肠道病毒基因组较小，短时间内即可复制出大量病毒进一步感染其他肿瘤细胞，引起强烈的细胞病变反应；再次，肠道病毒不含致癌基因，因此不会诱发肿瘤；最后，肠道病毒的基因组能够通过反向遗传学技术被改造，从而实现对病毒的减毒，减少其副作用。目前已有报导的具有溶瘤活性的肠道病毒包括用于治疗恶性神经胶质瘤等人实体肿瘤的嵌合脊髓灰质炎病毒(dobrikova等人，molther2008,16(11):1865-1872)；杀伤人黑色素瘤细胞的柯萨奇病毒a13、a15、a18和a21(au等人，virolj2011,8:22)；杀伤人胃癌和卵巢癌细胞的埃可病毒echo1(shafren等人，intjcancer2005,115(2):320-328；haley等人，jmolmed(berl)2009,87(4):385-399)等等。然而获得兼具肿瘤特异性和肿瘤杀伤活性的病毒仍然是有必要的。肠道病毒68型(ev-d68)是小rna病毒科肠道病毒属d种肠道病毒中的一种，其1962年首次从加利福尼亚呼吸道感染儿童分离得到(schieble等人，amjepidemiol1967,85(2):297-310)。与大多数肠道病毒耐酸、在人类胃肠道中繁殖的特性不同，ev-d68对酸敏感并且主要在呼吸道中复制。由于长期以来很少有ev-d68感染的病例报导，ev-d68被认为是一种罕见的病原体，主要引起轻度呼吸道疾病，包括流鼻涕、打喷嚏、咳嗽等。目前本领域尚未有任何肠道病毒68型具有溶瘤活性的报道。技术实现要素：在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、生物化学、细胞生物学、核酸化学等操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。如本文中所使用的，术语“肠道病毒68型(ev-d68)”是指，小rna病毒科肠道病毒属d种肠道病毒中的一种，其基因组为单股正链rna，由5’非编码区(5’utr)、一个开放阅读框(orf)、3’非编码区(3’utr)和多聚腺苷酸尾巴组成；其orf编码一个前体多聚蛋白，经过自身蛋白酶水解切割，可产生结构蛋白vp1～vp4和非结构蛋白2a、2b、2c、3a、3b、3c和3d；为了更加清楚地描述本发明，ev-d68基因组中上述各蛋白所对应的核酸序列分别称为vp1基因、vp2基因、vp3基因、vp4基因、2a基因、2b基因、2c基因、3a基因、3b基因、3c基因和3d基因。在本发明中，表述“肠道病毒68型(ev-d68)”意指野生型ev-d68，其可从自然中的来源分离并且没有被人为有意地修饰，其实例包括但不限于原型株ay426531(ca62-1)和ay426488(ca62-3)，以及各种临床分离株(例如，本发明实施例1中所描述的临床分离株)。野生型ev-d68的基因组序列或cdna序列是本领域熟知的，并且可参见各种公共数据库(例如，genbank数据库登录号km881710)。如本文中所使用的，术语病毒的“修饰形式”是指，对野生型病毒进行修饰而获得的经修饰的病毒，其保留了野生型病毒的期望的活性(例如，溶瘤活性)。在本发明中，ev-d68的“修饰形式”包括但不限于经修饰的ev-d68病毒，其与野生型ev-d68的基因组序列相比具有一个或多个核苷酸的替换、插入或删除，并且至少保留了ev-d68的溶瘤活性。如本文中所使用的，术语“溶瘤病毒”是指，能够感染肿瘤细胞，在肿瘤细胞中复制，引起肿瘤细胞死亡、裂解或阻止肿瘤细胞生长的病毒。优选地，该病毒对非肿瘤细胞具有最小的毒性效应。如本文中所使用的，术语“肿瘤特异性”是指，选择性地在肿瘤细胞内展现出生物功能或活性。例如，在本发明中，当术语“肿瘤特异性”用于描述病毒的杀伤选择性时，意指该病毒能够选择性杀伤肿瘤细胞而不杀伤或基本上不杀伤非肿瘤细胞，或者，该病毒对肿瘤细胞的杀伤比对非肿瘤细胞的杀伤更为有效。如本文中所使用的，术语“溶瘤活性”主要包括肿瘤杀伤活性。当描述病毒的溶瘤活性时，通常可以通过其病毒感染肿瘤细胞的能力、在肿瘤细胞内复制的能力和/或杀伤肿瘤细胞的能力等指标衡量该病毒的溶瘤活性。病毒的溶瘤活性可以采用本领域已知的任何方法进行测定。例如，病毒感染肿瘤细胞的能力可以通过测量感染给定百分率的肿瘤细胞(例如50％细胞)所需要的病毒剂量进行评价；在肿瘤细胞内复制的能力可以通过测量病毒在肿瘤细胞内的生长情况进行评价；杀伤肿瘤细胞的能力可以通过观察致细胞病变效应(cpe)或测量肿瘤细胞活性进行评价。如本文中所使用的，表述“ev-d68的cdna序列”意指，该病毒基因组rna序列的dna表示形式，其与所述rna序列相比差异仅在于所述rna序列中的核糖核苷酸被对应的脱氧核糖核苷酸替代，例如尿嘧啶核糖核苷酸(ump)由胸腺嘧啶脱氧核糖核苷酸(dtmp)替代。如本文中所使用的，术语“外源核酸”是指，人工引入的核苷酸序列，其相对于原始序列而言是外来的。外源核酸包括但不限于，未在所述病毒基因组中发现的任何基因或核苷酸序列。然而，在本发明中，特别优选地，所述外源核酸由至多1500个，例如至多1200个，至多1000个核苷酸组成。在某些情况下，优选地，外源核酸编码具有抗肿瘤杀伤活性的蛋白或多肽，例如细胞因子、或抗肿瘤蛋白或多肽；或者，外源核酸包含微小rna(microrna，mirna)的靶序列。在本发明中，所述微小rna优选为那些在肿瘤细胞中的表达量显著低于正常细胞和/或具有明显的组织特异性的微小rna，其实例包括但不限于，肝组织特异性表达的mir-122，mir-192，mir-483等；心脏特异性表达的mir-1，mir-133a/b，mir-208等；肾组织特异性表达的mir-192，mir-196a/b，mir-204，mir-215等；肌肉组织特异性表达的mir-133a/b，mir-206等；脑组织特异性表达的mir-124a，mir-125a/b，mir-128a/b，mir-138等；以及在肝肿瘤组织内低表达的mir-34，mir-122a，mir-26a；在肾肿瘤组织内低表达的mir-34；在膀胱肿瘤组织内低表达的mir-143，mir-133a/b；在肺肿瘤组织内低表达的mir-let-7，mir-29，等等(参见，例如，ruizajandrussellsj.micrornasandoncolyticviruses.[j].curropinvirol,2015,13:40–48；其全部通过引用并入本文)。在本发明中，当经修饰的ev-d68包含上述微小rna的靶序列时，其在该microrna高表达或特异性表达的细胞/组织中，受到该microrna的调控，从而可使该溶瘤病毒的复制减弱、甚至丧失杀伤活性，而在该microrna低表达或不表达的肿瘤细胞/组织中则正常复制、正常杀伤肿瘤细胞。如本文中所使用的，术语“细胞因子”具有本领域技术人员公知的含义。然而，在本发明中，当使用本发明的溶瘤病毒来治疗肿瘤时，特别优选地，所述细胞因子为能够用于肿瘤治疗的细胞因子。“细胞因子”的实例包括但不限于，白介素(例如il-2、il-12和il-15)、干扰素(例如ifnα、ifnβ、ifnγ)、肿瘤坏死因子(例如tnfα)、集落刺激因子(例如gm-csf)，及其任何组合(参见例如，ardolinom,hsuj,rauletdh.cytokinetreatmentincancerimmunotherapy[j].oncotarget,2015,6(23):19346-19347)。如本文中所使用的，术语“抗肿瘤蛋白或多肽”是指，具有治疗肿瘤活性的蛋白或多肽，其包括但不限于：(1)对细胞具有毒性、可抑制细胞增殖或诱导细胞凋亡的蛋白或多肽，其实例包括但不限于胸苷激酶tk(tk/gcv)、trail和fasl(参见例如，candolfim,kinggd,muhammadag,etal.evaluationofproapototictransgenestouseincombinationwithflt3linanimmune-stimulatorygenetherapyapproachforglioblastomamultiforme(gbm)[j].fasebj,2008,22:1077.13)；(2)具有免疫治疗作用的蛋白或多肽，其实例包括但不限于抗细胞毒t淋巴细胞相关抗原4(anti-ctla-4)、抗程序性死亡受体1(anti-pd-1)的单链抗体(scfv)和抗程序性死亡配体1(anti-pdl-1)的单链抗体(scfv)(参见例如，nolane,savasp,polichenian,etal.combinedimmunecheckpointblockadeasatherapeuticstrategyforbrca1-mutatedbreastcancer[j].sciencetransmed,2017,9:eaal4922；其全部通过引用并入本文)；(3)抑制肿瘤血管生成的蛋白或多肽，其实例包括但不限于抗血管内皮生长因子(anti-vegf)的单链抗体(scfv)、vegf来源多肽(例如d(lpr)、ksvrgkgkgqkrkrkksryk等)和atn-161(参见例如，roscaev，koskimakije，riveracg，etal.anti-angiogenicpeptidesforcancertherapeutics[j].currpharmbiotechnol，2011,12(8):1101–1116；其全部通过引用并入本文)。如本文中所使用的，术语“scfv”是指，包含重链可变区(vh)和轻链可变区(vl)的单个多肽链，其中所述vl和vh通过接头(linker)相连(参见，例如,bird等人,science242:423-426(1988)；huston等人,proc.natl.acad.sci.usa85:5879-5883(1988)；和pluckthun，thepharmacologyofmonoclonalantibodies，第113卷，roseburg和moore编，springer-verlag，纽约，第269-315页(1994))。此类scfv分子可具有一般结构：nh2-vl-接头-vh-cooh或nh2-vh-接头-vl-cooh。如本文中所使用的，术语“同一性”用于指两个蛋白/多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个dna分子的每一个中的某个位置都被腺嘌呤占据，或两个蛋白/多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，dna序列ctgact和caggtt共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如align程序(dnastar,inc.)方便地进行的needleman等人(1970)j.mol.biol.48：443-453的方法来实现。还可使用已整合入align程序(版本2.0)的e.meyers和w.miller(comput.applbiosci.，4:11-17(1988))的算法，使用pam120权重残基表(weightresiduetable)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入gcg软件包(可在www.gcg.com上获得)的gap程序中的needleman和wunsch(jmoibiol.48:444-453(1970))算法，使用blossum62矩阵或pam250矩阵以及16、14、12、10、8、6或4的缺口权重(gapweight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(yac)、细菌人工染色体(bac)或p1来源的人工染色体(pac)；噬菌体如λ噬菌体或m13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如sv40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。如本文中所使用的，术语“内部核糖体进入位点(ires)”是指，一段位于信使rna(mrna)序列中的核苷酸序列，其能够起始翻译而不依赖于5'帽结构。ires通常位于5'非翻译区(5'utr)中，但也可能位于在mrna的其他位置。如本文中所使用的，术语“人鼻病毒2(hrv2)”是指，小rna病毒科的一种病毒，其基因组序列或cdna序列是本领域公知的，可参见各种公共数据库(例如，genbank数据库登录号x02316.1)。如本文中所使用的，表述“包含ev-d68或其修饰形式的基因组序列的核酸分子”或“核酸分子包含ev-d68或其修饰形式的基因组序列”具有本领域技术人员通常理解的含义，即当所述核酸分子为dna时，所述核酸分子包含ev-d68或其修饰形式的基因组序列的dna表示形式；当所述核酸分子为rna时，所述核酸分子包含ev-d68或其修饰形式的基因组序列。如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指，在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如remington'spharmaceuticalsciences.editedbygennaroar,19thed.pennsylvania:mackpublishingcompany,1995)，并且包括但不限于：ph调节剂，表面活性剂，离子强度增强剂，维持渗透压的试剂，延迟吸收的试剂，稀释剂，佐剂，防腐剂，稳定剂等。例如，ph调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如tween-80。离子强度增强剂包括但不限于氯化钠。维持渗透压的试剂包括但不限于糖、nacl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水，水性缓冲液(如缓冲盐水)，醇和多元醇(如甘油)等。佐剂包括但不限于铝佐剂(例如氢氧化铝)，弗氏佐剂(例如完全弗氏佐剂)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如硫柳汞，2-苯氧乙醇，对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。稳定剂具有本领域技术人员通常理解的含义，其能够稳定药物中的活性成分的期望活性(例如溶瘤活性)，包括但不限于谷氨酸钠，明胶，spga，糖类(如山梨醇，甘露醇，淀粉，蔗糖，乳糖，葡聚糖，或葡萄糖)，氨基酸(如谷氨酸，甘氨酸)，蛋白质(如干燥乳清，白蛋白或酪蛋白)或其降解产物(如乳白蛋白水解物)等。如本文中所使用的，术语“治疗”是指，治疗或治愈疾病(例如肿瘤)，延缓疾病(例如肿瘤)的症状的发作，和/或延缓疾病(例如肿瘤)的发展。如本文中所使用的，术语“有效量”是指，可以有效实现预期目的的量。例如，治疗有效量可以是有效地或足以治疗或治愈疾病(例如肿瘤)，延缓疾病(例如肿瘤)症状的发作和/或延缓疾病(例如肿瘤)发展的量。这样的有效量可以由本领域技术人员或医生容易地确定，并且可以与预期目的(例如治疗)、受试者的一般健康状况、年龄、性别、体重、待治疗的疾病的严重程度、并发症、施用方式等相关。这样的有效量的确定完全在本领域技术人员的能力范围内。如本文中使用的，术语“受试者”是指哺乳动物，例如灵长类哺乳动物，例如人。在某些实施方式中，所述受试者(例如人)患有肿瘤，或者，具有患有肿瘤的风险。本申请的发明人经过大量实验和反复摸索，出人意料地发现，肠道病毒68型具有广谱、显著的肿瘤细胞杀伤能力。基于这一发现，本发明人开发了新的用于治疗肿瘤的溶瘤病毒以及基于该病毒的肿瘤治疗方法。因此，在一个方面，本发明提供了肠道病毒68型(ev-d68)或其修饰形式，或一种核酸分子，用于在受试者中治疗肿瘤的用途，或者用于制备在受试者中治疗肿瘤的药物的用途；其中，所述核酸分子包含选自下列的序列：(1)ev-d68或其修饰形式的基因组序列或cdna序列；和(2)所述基因组序列或cdna序列的互补序列。在某些优选的实施方案中，所述ev-d68为野生型ev-d68。在某些优选的实施方案中，所述ev-d68可以是从感染肠道病毒68型的个体中分离得到的临床分离毒株。在某些优选的实施方案中，所述ev-d68或其修饰形式的基因组序列与如seqidno：12所示的核苷酸序列具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。在某些优选的实施方案中，所述ev-d68或其修饰形式的基因组序列为如seqidno：12所示的核苷酸序列。在某些优选的实施方案中，所述ev-d68或其修饰形式的cdna序列与如seqidno：1所示的核苷酸序列具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。在某些优选的实施方案中，所述ev-d68或其修饰形式的cdna序列为如seqidno：1所示的核苷酸序列。在某些优选的实施方案中，所述修饰形式为经修饰的ev-d68，其与野生型ev-d68相比在基因组中具有一个或多个核苷酸的替换、插入或删除。在某些优选的实施方案中，所述经修饰的ev-d68与野生型ev-d68相比，具有一个或多个选自下列的修饰：(1)位于非翻译区(例如5’utr或3’utr)的一个或多个突变；(2)一个或多个外源核酸的插入；(3)一个或多个内源基因的缺失或突变；和(4)上述三项的任意组合。在某些优选的实施方案中，所述经修饰的ev-d68包括位于5’非翻译区(5’utr)的一个或多个突变。在某些优选的实施方案中，所述经修饰的ev-d68的5’utr序列经全部或部分替换。在某些优选的实施方案中，所述经修饰的ev-d68的5’utr中的内部核糖体进入位点(ires)序列替换为外源ires序列，例如人鼻病毒2(hrv2)的内部核糖体进入位点序列。在某些优选的实施方案中，所述人鼻病毒2(hrv2)的内部核糖体进入位点序列如seqidno：2所示。人鼻病毒2(hrv2)的内部核糖体进入位点序列的使用在某些情况下是有利的，例如有益于提高溶瘤病毒的肿瘤特异性。之前已报道，在正常人神经细胞中，人鼻病毒2的内部核糖体进入位点序列会被宿主rna结合蛋白(drbp76和nf45)特异结合，从而阻止了elf4g等因子的招募(merrill等人，jvirol2006,80(7):3147-3156；merrill和gromeier，jvirol2006,80(14):6936-6942；neplioueva等人，plosone2010,5(7):e11710)；同时，由于缺乏raf/erk1/2/mapk等信号通路的支持，核糖体难以结合到人鼻病毒2的内部核糖体进入位点序列上，因此无法启动病毒蛋白翻译(dobrikov等人，molcellbiol2011,31(14):2947-2959；dobrikov等人，molcellbiol2013,33(5):937-946)。在人神经胶质瘤肿瘤细胞中，人鼻病毒2的内部核糖体进入位点却不受以上两个因素影响，可正常启动病毒蛋白转录与翻译。因此，在某些情况下，将ev-d68的内部核糖体进入位点序列替换为人鼻病毒2的内部核糖体进入位点序列有益于避免或减轻本发明所述病毒对正常人神经细胞产生毒副作用，而不影响该病毒用于人神经胶质瘤的治疗。在某些优选的实施方案中，所述经修饰的ev-d68包含外源核酸。在某些优选的实施方案中，所述外源核酸编码细胞因子(例如gm-csf，优选为人gm-csf)、或抗肿瘤蛋白或多肽(例如，抗pd-1或pd-l1的scfv，优选为抗人pd-1或pd-l1的scfv)。在某些优选的实施方案中，所述外源核酸的插入位点位于所述经修饰的ev-d68基因组的5’utr与vp4基因之间，或者位于vp1基因与2a基因之间。在某些优选的实施方案中，所述外源核酸包含微小rna(microrna,mirna)(例如mir-133或mir-206)的靶序列。在某些优选的实施方案中，所述微小rna的靶序列的插入位点在所述经修饰的ev-d68基因组的3’非翻译区(3’utr)。之前已报道，某些微小rna在肿瘤细胞中的表达量显著低于正常细胞和/或具有明显的组织特异性，因此在某些情况下，本发明的经修饰的ev-d68包含这类微小rna的靶序列是有利的，因为在正常细胞或组织内高表达的这类微小rna可以通过对应的靶序列减少甚至阻断经修饰的ev-d68在该正常细胞或组织内的复制，从而减轻甚至避免经修饰的ev-d68对非肿瘤细胞的毒副作用。这类微小rna包括但不限于mir-133、mir-206、mir-1、mir-143、mir-145、mir-217、let-7、mir-15、mir-16等(参见例如，pct国际申请wo2008103755a1，美国专利申请us20160143969a1，或baohongzhang等人，developmentalbiology，volume302,issue1,1february2007,pages1–12；其全部通过引用并入本文)。在某些优选的实施方案中，所述外源核酸包括一个或多个(例如2个，3个或4个)如上所述的微小rna的靶序列。在某些优选的实施方案中，所述外源核酸包含mir-133和/或mir-206的靶序列。在某些优选的实施方案中，所述mir-133的靶序列如seqidno:3所示。在某些优选的实施方案中，所述mir-206的靶序列如seqidno:4所示。在某些情况下，mir-133和/或mir-206的靶序列的插入是有利的。这是由于mir-133和mir-206在肌肉组织中特异性表达，因此通过将mir-133和/或mir-206的靶序列插入所述经修饰的ev-d68中的方式可以改变该溶瘤病毒的组织嗜性，以减小或避免对正常肌肉组织的杀伤。在某些优选的实施方案中，所述经修饰的ev-d68包含至少一种如上所述的外源核酸的插入和/或至少一种如上所述的位于非翻译区的突变。在某些优选的实施方案中，所述经修饰的ev-d68的基因组序列与选自下列的核苷酸序列具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性：如seqidno：13-16所示的核苷酸序列。在某些优选的实施方案中，所述经修饰的ev-d68的基因组序列选自如seqidno：13-16任一项所示的核苷酸序列。在某些优选的实施方案中，所述经修饰的ev-d68的cdna序列与选自下列的核苷酸序列具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性：如seqidno：8-11所示的核苷酸序列。在某些优选的实施方案中，所述经修饰的ev-d68的cdna序列选自如seqidno：8-11任一项所示的核苷酸序列。在本发明中，所述经修饰的ev-d68可以通过反向遗传学技术获得，所述反向遗传学技术是本领域已知的，例如可参见yangls，lisx，liuyj，etal.virusres，2015,210:165-168；houwh,yangls，lisx，etal.virusres，2015,205:41-44；其全部通过引用并入本文。在此类实施方案中，通常对野生型ev-d68的cdna进行修饰(例如，外源核酸的插入、内源基因的缺失或突变或位于非翻译区的突变)从而获得经修饰的ev-d68。在本发明中，所述ev-d68或其修饰形式可以经预处理以减少或消除受试者对该病毒的免疫反应，其中所述预处理可以包括：将所述ev-d68包装在脂质体或胶束中，和/或使用蛋白酶(例如，糜蛋白酶或胰蛋白酶)去除病毒的衣壳蛋白以减小宿主对病毒的体液和/或细胞免疫。在本发明中，可以将所述ev-d68或其修饰形式在肿瘤细胞内连续适应性传代。在某些优选的实施方案中，所述肿瘤细胞可以为本领域已知的肿瘤细胞系或肿瘤细胞株，也可以为从患有肿瘤的个体(例如，受试者)体内经手术切除或临床分离而得到的肿瘤细胞。在某些优选的实施方案中，将所述ev-d68或其修饰形式在获得自患有肿瘤的个体(例如，受试者)体内的肿瘤细胞内连续适应性传代。在某些优选的实施方案中，所述肿瘤细胞从患有肿瘤的个体(例如，受试者)体内经手术切除或临床分离而得到。在某些优选的实施方案中，所述连续适应性传代的方法包括多个(例如至少5个，至少10个，至少15个，至少20个)由下述过程所构成的周期：1)将病毒感染靶肿瘤细胞；2)收获上清中的病毒；和3)将获得的病毒重新感染新鲜靶肿瘤细胞。在某些优选的实施方案中，可以组合使用如上所述的ev-d68和其修饰形式。因此，所述药物可以包括ev-d68以及其修饰形式中的一种或数种。在某些优选的实施方案中，所述核酸分子由如本文所述的ev-d68或其修饰形式的基因组序列或cdna序列，或所述基因组序列或cdna序列的互补序列组成。在某些优选的实施方案中，所述核酸分子具有如本文所述的ev-d68或其修饰形式的基因组序列。在某些优选的实施方案中，所述核酸分子为rna。在某些优选的实施方案中，所述核酸分子具有如seqidno:12-16任一项所示的核苷酸序列。在某些优选的实施方案中，所述核酸分子为包含如本文所述的ev-d68或其修饰形式的基因组序列或cdna序列，或所述基因组序列或cdna序列的互补序列的载体(例如，克隆载体或表达载体)。在某些优选的实施方案中，所述核酸分子为包含如本文所述的ev-d68或其修饰形式的cdna序列，或所述cdna序列的互补序列的载体(例如，克隆载体或表达载体)。在某些优选的实施方案中，所述核酸分子包含ev-d68或其修饰形式的基因组序列的互补序列。在某些优选的实施方案中，所述互补序列与选自下列的核苷酸序列互补：(1)如seqidno：12所示的核苷酸序列；(2)与如seqidno：12所示的核苷酸序列具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的核苷酸序列；(3)如seqidno：13-16任一项所示的核苷酸序列；和(4)与如seqidno：13-16任一项所示的核苷酸序列具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的核苷酸序列。在某些优选的实施方案中，所述核酸分子包含ev-d68或其修饰形式的cdna序列的互补序列。在某些优选的实施方案中，所述互补序列与选自下列的核苷酸序列互补：(1)如seqidno：1所示的核苷酸序列；(2)与如seqidno：1所示的核苷酸序列具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的核苷酸序列；(3)如seqidno：8-11任一项所示的核苷酸序列；和(4)与如seqidno：8-11任一项所示的核苷酸序列具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的核苷酸序列。在本发明中，所述核酸分子可以通过本领域已知的任何方式进行递送，例如，直接注射裸露的核酸分子(例如，裸rna)，或利用非病毒递送系统(non-viraldeliverysystem)。所述非病毒递送系统可通过本领域熟知的各种材料制备获得，其中所述材料包括但不限于详细描述于“yinh,etal.natrevgenet.2014aug；15(8):541-55.”以及“rileymk,vermerrisw.nanomaterials(basel).2017apr28；7(5).pii:e94.”中的各种材料，其全部通过引用并入本文，例如脂质体、无机纳米粒子(如金纳米颗粒)、多聚物(如peg)等等。在某些优选的实施方案中，所述药物包含治疗有效量的ev-d68和/或其修饰形式，或治疗有效量的如本文所述的核酸分子。在某些优选的实施方案中，所述药物可以为医学领域已知的任何形式。例如，所述药物可以是片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、冻干粉剂)等形式。在一些实施方案中，所述药物为注射液或冻干粉剂。在某些优选的实施方案中，所述药物还包含药学上可接受的载体或赋形剂。在某些优选的实施方案中，所述药物包含稳定剂。在某些优选的实施方案中，所述药物任选地还包含另外的药学活性剂。在一个优选的实施方案中，所述另外的药学活性剂是具有抗肿瘤活性的药物，例如另外的溶瘤病毒、化学治疗剂或免疫治疗剂。在本发明中，所述另外的溶瘤病毒包括但不限于疱疹病毒、腺病毒、细小病毒、呼肠孤病毒、新城疫病毒、水疱性口炎病毒、麻疹病毒或其任意组合。所述化学治疗剂包括但不限于5-氟尿嘧啶、丝裂霉素、甲氨蝶呤、羟基脲、环磷酰胺、达卡巴嗪、米托蒽醌、蒽环类(如表柔比星或多柔比星)、依托泊苷、铂类化合物(如卡铂或顺铂)、紫杉烷类(如紫杉醇或紫杉特尔)或其任意组合。所述免疫治疗剂包括但不限于免疫卡控点抑制剂(如pd-l1/pd-1抑制剂或ctla-4抑制剂)、肿瘤特异性靶向抗体(如利妥昔单抗或赫赛汀)或其任意组合。在某些优选的实施方案中，所述药物包含单位剂量的ev-d68和/或其修饰形式，例如包含至少1x102pfu、至少1x103pfu、至少1x104pfu、1x105pfu、1x106pfu、至少1x107pfu、至少1x108pfu、至少1x109pfu、至少1x1010pfu、至少1x1011pfu、至少1x1012pfu、至少1x1013pfu、至少1x1014pfu或至少1x1016pfu的ev-d68和/或其修饰形式。在某些优选的实施方案中，所述药物包含1x102pfu～1x1017pfu的ev-d68和/或其修饰形式。在某些优选的实施方案中，所述药物含有单位剂量的如本文所述的核酸分子，例如含有3xl010～3xlo14病毒基因组拷贝数(virusgenomecopies)的所述核酸分子。在某些优选的实施方案中，可以将所述药物与额外的疗法一起施用。这种额外的疗法可以是已知用于肿瘤的任何疗法，例如手术、化学治疗、放射治疗、免疫治疗、激素治疗或基因治疗。这种额外的疗法可以在施用所述药物之前、同时或之后施用。在某些优选的实施方案中，所述肿瘤包括但不限于，宫颈癌、卵巢癌、子宫内膜癌、肺癌、肝癌、肾癌、神经母细胞瘤、神经胶质瘤、乳腺癌、黑色素瘤、前列腺癌、膀胱癌、胰腺癌、胃癌、结直肠癌、食管癌、甲状腺癌、喉癌、成骨肉瘤、造血系统恶性肿瘤(如淋巴瘤或白血病)。在某些优选的实施方案中，所述受试者为哺乳动物，例如人。在另一个方面，本发明提供了一种治疗肿瘤的方法，其包括向有此需要的受试者施用有效量的ev-d68或其修饰形式，或有效量的核酸分子的步骤；其中，所述核酸分子包含选自下列的序列：(1)ev-d68或其修饰形式的基因组序列或cdna序列；和(2)所述基因组序列或cdna序列的互补序列。在某些优选的实施方案中，向所述受试者施用ev-d68。在某些优选的实施方案中，所述ev-d68为野生型ev-d68。在某些优选的实施方案中，所述ev-d68可以是从感染肠道病毒68型的个体中分离得到的临床分离毒株。在某些优选的实施方案中，所述ev-d68或其修饰形式的基因组序列与如seqidno：12所示的核苷酸序列具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。在某些优选的实施方案中，所述ev-d68或其修饰形式的基因组序列为如seqidno：12所示的核苷酸序列。在某些优选的实施方案中，所述ev-d68或其修饰形式的cdna序列与如seqidno：1所示的核苷酸序列具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。在某些优选的实施方案中，所述ev-d68或其修饰形式的cdna序列为如seqidno：1所示的核苷酸序列。在某些优选的实施方案中，向所述受试者施用ev-d68的修饰形式。在某些优选的实施方案中，所述修饰形式为经修饰的ev-d68，其与野生型ev-d68相比在基因组中具有一个或多个核苷酸的替换、插入或删除。在某些优选的实施方案中，所述经修饰的ev-d68与野生型ev-d68相比，具有一个或多个选自下列的修饰：(1)位于非翻译区(例如，5’utr或3’utr)的一个或多个突变；(2)一个或多个外源核酸的插入；(3)一个或多个内源基因的缺失或突变；和(4)上述三项的任意组合。在某些优选的实施方案中，所述经修饰的ev-d68包括位于5’非翻译区(5’utr)的一个或多个突变。在某些优选的实施方案中，所述经修饰的ev-d68的5’utr序列经全部或部分替换。在某些优选的实施方案中，所述经修饰的ev-d68的5’utr中的内部核糖体进入位点(ires)序列替换为外源ires序列，例如人鼻病毒2(hrv2)的内部核糖体进入位点序列。在某些优选的实施方案中，所述人鼻病毒2(hrv2)的内部核糖体进入位点序列如seqidno：2所示。在某些优选的实施方案中，所述经修饰的ev-d68包含外源核酸。在某些优选的实施方案中，所述外源核酸编码细胞因子(例如gm-csf，优选为人gm-csf)、或抗肿瘤蛋白或多肽(例如抗pd-1或pd-l1的scfv，优选为抗人pd-1或pd-l1的scfv)。在某些优选的实施方案中，所述外源核酸的插入位点位于所述经修饰的ev-d68基因组的5’utr与vp4基因之间，或者位于vp1基因与2a基因之间。在某些优选的实施方案中，所述外源核酸包含微小rna(microrna,mirna)(例如mir-133或mir-206)的靶序列。在某些优选的实施方案中，所述微小rna的靶序列的插入位点在所述经修饰的ev-d68基因组的3’非翻译区(3’utr)。在某些优选的实施方案中，所述外源核酸包括一个或多个(例如2个，3个或4个)如上所述的微小rna的靶序列。在某些优选的实施方案中，所述外源核酸包含mir-133和/或mir-206的靶序列。在某些优选的实施方案中，所述mir-133的靶序列如seqidno:3所示。在某些优选的实施方案中，所述mir-206的靶序列如seqidno:4所示。在某些优选的实施方案中，所述经修饰的ev-d68包含至少一种如上所述的外源核酸的插入和/或至少一种如上所述的位于非翻译区的突变。在某些优选的实施方案中，所述经修饰的ev-d68的基因组序列与选自下列的核苷酸序列具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性：如seqidno：13-16所示的核苷酸序列。在某些优选的实施方案中，所述经修饰的ev-d68的基因组序列选自如seqidno：13-16任一项所示的核苷酸序列。在某些优选的实施方案中，所述经修饰的ev-d68的cdna序列与选自下列的核苷酸序列具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性：如seqidno：8-11所示的核苷酸序列。在某些优选的实施方案中，所述经修饰的ev-d68的cdna序列选自如seqidno：8-11任一项所示的核苷酸序列。在某些优选的实施方案中，可以组合使用如上所述的ev-d68和其修饰形式。因此，可以向受试者施用ev-d68以及其修饰形式中的一种或多种。在某些优选的实施方案中，向所述受试者施用如本文所述的核酸分子。在某些优选的实施方案中，所述核酸分子由如本文所述的ev-d68或其修饰形式的基因组序列或cdna序列，或所述基因组序列或cdna序列的互补序列组成。在某些优选的实施方案中，所述核酸分子具有如本文所述的ev-d68或其修饰形式的基因组序列。在某些优选的实施方案中，所述核酸分子为rna。在某些优选的实施方案中，所述核酸分子具有如seqidno:12-16任一项所示的核苷酸序列。在某些优选的实施方案中，所述核酸分子为包含如本文所述的ev-d68或其修饰形式的基因组序列或cdna序列，或所述基因组序列或cdna序列的互补序列的载体(例如，克隆载体或表达载体)。在某些优选的实施方案中，所述核酸分子为包含如本文所述的ev-d68或其修饰形式的cdna序列，或所述cdna序列的互补序列的载体(例如，克隆载体或表达载体)。在某些优选的实施方案中，所述核酸分子包含ev-d68或其修饰形式的基因组序列的互补序列。在某些优选的实施方案中，所述互补序列与选自下列的核苷酸序列互补：(1)如seqidno：12所示的核苷酸序列；(2)与如seqidno：12所示的核苷酸序列具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的核苷酸序列；(3)如seqidno：13-16任一项所示的核苷酸序列；和(4)与如seqidno：13-16任一项所示的核苷酸序列具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的核苷酸序列。在某些优选的实施方案中，所述核酸分子包含ev-d68或其修饰形式的cdna序列的互补序列。在某些优选的实施方案中，所述互补序列与选自下列的核苷酸序列互补：(1)如seqidno：1所示的核苷酸序列；(2)与如seqidno：1所示的核苷酸序列具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的核苷酸序列；(3)如seqidno：8-11任一项所示的核苷酸序列；和(4)与如seqidno：8-11任一项所示的核苷酸序列具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的核苷酸序列。在本发明中，可以通过本领域已知的任何方式递送所述核酸分子，例如，直接注射裸露的核酸分子(例如，裸rna)，或利用非病毒递送系统(non-viraldeliverysystem)。所述非病毒递送系统可通过本领域熟知的各种材料制备获得，其中所述材料包括但不限于详细描述于“yinh,etal.natrevgenet.2014aug；15(8):541-55.”以及“rileymk,vermerrisw.nanomaterials(basel).2017apr28；7(5).pii:e94.”中的各种纳米材料，其全部通过引用并入本文，例如脂质体、无机纳米粒子(如金纳米颗粒)、多聚物(如peg)等等。在某些优选的实施方案中，可以将ev-d68和/或其修饰形式，或如本文所述的核酸分子，作为药物组合物进行配制和施用。这样的药物组合物可包含治疗有效量的ev-d68和/或其修饰形式，或治疗有效量的如本文所述的核酸分子。在某些优选的实施方案中，所述药物组合物可以是医学领域已知的任何形式。例如，所述药物组合物可以是片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、冻干粉剂)等形式。在一些实施方案中，所述药物组合物为注射液或冻干粉剂。在某些优选的实施方案中，所述药物组合物还包含药学上可接受的载体或赋形剂。在某些优选的实施方案中，所述药物组合物包含稳定剂。在本发明中可以通过各种合适的方式，向受试者施用ev-d68和/或其修饰形式，或如本文所述的核酸分子。在某些情况下，ev-d68和/或其修饰形式，或如本文所述的核酸分子的施用方式取决于肿瘤的位置和类型。例如，对于容易接近的实体肿瘤，任选地通过直接向肿瘤注射(例如，瘤内注射)来施用该病毒或核酸分子；对于造血系统的肿瘤，可以通过静脉内或其他血管内途径施用该病毒或核酸分子；对于体内不容易接近的肿瘤(例如转移瘤)，可以系统地施用该病毒或核酸分子以使其遍布全身并由此到达肿瘤(例如，静脉内或肌肉内注射)。任选地，可以经皮下、腹膜内、鞘内(例如对于脑部肿瘤)、局部(例如对于黑素瘤)、口服(例如对于口腔或食道癌)、经鼻或通过吸入喷雾(例如对于肺癌)等途径施用本发明的病毒或核酸分子。在某些优选的实施方案中，可以通过皮内注射、皮下注射、肌肉注射、静脉注射、口服给予等途径施用本发明的ev-d68和/或其修饰形式，或如本文所述的核酸分子。在某些优选的实施方案中，所述方法还包括施用具有抗肿瘤活性的另外的药学活性剂。这种另外的药学活性剂可以在施用ev-d68和/或其修饰形式，或如本文所述的核酸分子之前、同时或之后施用。在某些优选的实施方案中，所述另外的药学活性剂包括另外的溶瘤病毒、化学治疗剂或免疫治疗剂。在本发明中，所述另外的溶瘤病毒包括但不限于疱疹病毒、腺病毒、细小病毒、呼肠孤病毒、新城疫病毒、水疱性口炎病毒、麻疹病毒或其任意组合。所述化学治疗剂包括但不限于5-氟尿嘧啶、丝裂霉素、甲氨蝶呤、羟基脲、环磷酰胺、达卡巴嗪、米托蒽醌、蒽环类(如表柔比星或多柔比星)、依托泊苷、铂类化合物(如卡铂或顺铂)、紫杉烷类(如紫杉醇或紫杉特尔)或其任意组合。所述免疫治疗剂包括但不限于免疫卡控点抑制剂(如pd-l1/pd-1抑制剂或ctla-4抑制剂)、肿瘤特异性靶向抗体(如利妥昔单抗或赫赛汀)或其任意组合。在某些优选的实施方案中，可以以1～1x1015pfu/kg受试者体重的任何量施用ev-d68和/或其修饰形式，例如以至少1x103pfu/kg、至少1x104pfu/kg、1x105pfu/kg、1x106pfu/kg、至少1x107pfu/kg、至少1x108pfu/kg、至少1x109pfu/kg、至少1x1010pfu/kg、至少1x1011pfu/kg或至少1x1012pfu/kg受试者体重的量施用ev-d68和/或其修饰形式。在某些优选的实施方案中，可以以3xl010～3xlo14病毒基因组拷贝数(virusgenomecopies)/kg受试者体重的任何量施用如本文所述的核酸分子。在某些优选的实施方案中，可以以每日3次、每日2次、每日1次、每两日1次或每周1次的方式施用ev-d68和/或其修饰形式，或如本文所述的核酸分子，任选地酌情每周或每月重复如上所述的给药方案。在某些优选的实施方案中，所述方法还包括施用额外的疗法。这种额外的疗法可以是已知用于肿瘤的任何疗法，例如手术、化学治疗、放射治疗、免疫治疗、激素治疗或基因治疗。这种额外的疗法可以在施用如上所述的方法之前、同时或之后施用。在某些优选的实施方案中，所述受试者是哺乳动物，例如人。在某些优选的实施方案中，所述肿瘤包括但不限于，宫颈癌、卵巢癌、子宫内膜癌、肺癌、肝癌、肾癌、神经母细胞瘤、神经胶质瘤、乳腺癌、黑色素瘤、前列腺癌、膀胱癌、胰腺癌、胃癌、结直肠癌、食管癌、甲状腺癌、喉癌、成骨肉瘤、造血系统恶性肿瘤(如淋巴瘤或白血病)。在另一个方面，本发明还涉及一种药物组合物，其包含如本文所述的ev-d68和/或其修饰形式，或如本文所述的核酸分子。在某些优选的实施方案中，所述药物组合物可以为医学领域已知的任何形式。例如，所述药物组合物可以是片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、冻干粉剂)等形式。在一些实施方案中，所述药物组合物为注射液或冻干粉剂。在某些优选的实施方案中，所述药物组合物还包含药学上可接受的载体或赋形剂。在某些优选的实施方案中，所述药物组合物包含稳定剂。在某些优选的实施方案中，所述药物组合物任选地还包含另外的药学活性剂。在一个优选的实施方案中，所述另外的药学活性剂是具有抗肿瘤活性的药物，例如另外的溶瘤病毒、化学治疗剂或免疫治疗剂。在某些优选的实施方案中，所述药物组合物用于在受试者中治疗肿瘤。在某些优选的实施方案中，所述受试者是哺乳动物，例如人。在某些优选的实施方案中，所述肿瘤包括但不限于，宫颈癌、卵巢癌、子宫内膜癌、肺癌、肝癌、肾癌、神经母细胞瘤、神经胶质瘤、乳腺癌、黑色素瘤、前列腺癌、膀胱癌、胰腺癌、胃癌、结直肠癌、食管癌、甲状腺癌、喉癌、成骨肉瘤、造血系统恶性肿瘤(如淋巴瘤或白血病)。在另一个方面，本发明还涉及如本文所述的ev-d68和/或其修饰形式，或如本文所述的核酸分子，其用作药物。发明的有益效果与现有技术相比，本发明的技术方案至少具有以下有益效果：本申请的发明人首次发现，肠道病毒68型(ev-d68)具有广谱肿瘤杀伤活性。基于这一发现，本发明进一步提供了基于ev-d68的溶瘤病毒，其具有更广谱的肿瘤杀伤活性以及更高的肿瘤特异性，特别是对造血系统恶性肿瘤(例如淋巴瘤或白血病)亦具有极高的杀伤能力，可单独用于肿瘤的治疗，亦可用作传统肿瘤治疗的辅助方法，或作为缺少其他治疗方法时的治疗手段。本发明的ev-d68或其修饰形式对正常细胞影响较小或无影响，并且不引发受试者(例如人)针对该病毒的免疫原性反应，从而可安全地施用给受试者(例如人)。因此，本发明的ev-d68或其修饰形式具有重大的临床价值。下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。附图说明图1显示了实施例2中野生型ev-d68对人脐静脉内皮细胞系huvec、人食管癌细胞系te-1、人甲状腺癌细胞系sw-579和tt的体外杀伤实验结果的显微镜照片，其中，mock表示未感染病毒的细胞。结果显示，在感染复数(moi)为10感染72小时后，ev-d68对人肿瘤细胞系te-1、sw-579和tt均有显著的溶瘤作用，但对人正常细胞的huvec无任何影响。图2显示了实施例2中野生型ev-d68对人肝癌细胞系hepg2、smmc7721、bel7404、bel7402和huh7；人宫颈癌细胞系hela和caski；人肺癌细胞系nci-h1299和a549；人包皮成纤维细胞系hff-1、人胚肾细胞系hek-293和分化后的人肝祖细胞系heparg的体外杀伤实验结果的结晶紫染色照片，其中，mock表示未感染病毒的细胞。结果显示，在moi为10、1和0.1感染72小时后，ev-d68对人肿瘤细胞系hepg2、smmc7721、bel7404、bel7402、huh7、hela、caski、nci-h1299和a549均有显著的溶瘤作用，但对人正常细胞的hff-1、hek-293和分化后的heparg影响很小。图3显示了实施例2中通过体外转录方法获得的同批4份野生型ev-d68病毒基因组rna样品的电泳图。图4显示了实施例2中野生型ev-d68病毒基因组rna对人宫颈癌肿瘤细胞系hela的杀伤效果。结果显示，转染ev-d68基因组rna的hela细胞在转染后24小时出现cpe已十分明显，到48小时几乎全部裂解死亡。图5a-5c显示了实施例3中野生型ev-d68对人宫颈癌细胞系hela(a)、人神经胶质瘤细胞系u118-mg(b)和人淋巴瘤细胞系raji(c)的体内抗肿瘤实验结果。结果显示，在攻毒实验组中，每隔2天瘤内注射106tcid50/每瘤块的ev-d68，共计5次后，分别皮下接种了hela、或u118-mg、或raji细胞的scid小鼠所形成的肿瘤的生长明显变慢、停滞，甚至裂解消失；相比之下，未经溶瘤病毒治疗的阴性组(ctrl)的肿瘤则保持正常生长，其肿瘤体积显著大于攻毒实验组。图6显示了实施例4中ev-d68-wt在balb/c小鼠中的毒性检测实验结果。图6a显示了1日龄balb/c小鼠在不同剂量(103、104、105、106和107tcid50/只)ev-d68腹腔攻毒后的小鼠存活率和健康分数；图6b显示了采用极高剂量(107tcid50/只)腹腔攻毒不同日龄(1日、2日、3日、7日和14日)balb/c小鼠后小鼠的存活率和健康分数。ev-d68对balb/c小鼠的整体毒性较弱，仅在使用高剂量时对1-3日龄balb/c小鼠致病致死，而对4日龄及以上balb/c小鼠无影响，表明ev-d68具有良好的体内安全性。序列信息本发明涉及的部分序列的信息提供于下面的表1中。表1：序列的描述具体实施方式现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。除非特别指明，本发明中所使用的分子生物学实验方法和免疫检测法，基本上参照j.sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及f.m.ausubel等人，精编分子生物学实验指南，第3版，johnwiley&sons，inc.，1995中所述的方法进行；限制性内切酶的使用依照产品制造商推荐的条件。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。本文中提及的全部公开案和其他参考资料以其全文通过引用合并入本文。实施例1：ev-d68及其修饰形式的获得及制备1.1从患者临床标本中分离肠道病毒ev-d68(1)患者的咽拭子来源于中国，厦门市疾病预防与控制中心；非洲绿猴肾细胞(vero细胞；number:ccl-81tm)由中国，厦门大学，国家传染病诊断试剂与疫苗工程技术研究中心保存，使用添加有10％胎牛血清以及谷氨酰胺、青霉素和链霉素的mem培养基进行培养。(2)标本处理：将患者咽拭子在标本保存液中充分搅动，以洗下拭子上粘附的病毒及含有病毒的细胞等，随后将标本保存液在4℃条件下4000rpm高速离心30min；(3)接种与观察：a.将vero细胞铺24孔板，每孔细胞数量为1×105个，吸去生长液(mem培养基，10％胎牛血清以及谷氨酰胺、青霉素和链霉素)，每孔换上1ml的维持液(mem培养基，2％胎牛血清以及谷氨酰胺、青霉素和链霉素)，随后除了阴性对照孔，每孔接种50μl的标本上清液，37℃，5％co2培养箱培养；b.一周内每天在显微镜下观察细胞，记录接种孔内是否有特征性的细胞病变效应(cpe)的出现；c.若7天内接种孔细胞出现肠道病毒特征性cpe时，收集细胞及上清至-80℃冻存；若7天后仍无cpe出现，将细胞进行盲传；d.盲传6代内出现细胞cpe的，收集细胞及上清至-80℃冻存；盲传6代后仍未出现cpe的，判定为阴性；(4)病毒的分离与克隆化：使用rt-pcr(piralla等人，jclinmicrobiol2015,53(5):1725-1726)与基于特异性抗体的酶联免疫斑点法(yang等人，clinvaccineimmunol2014,21(3):312-320；hou等人，jvirolmethods2015,215-216:56-60)鉴定从临床标本中分离获得的病毒，选取ev-d68阳性的培养物进行至少3次克隆化实验；对每一轮由有限稀释法获得的克隆毒株同样使用rt-pcr与elispot鉴定，选取ev-d68阳性的克隆毒株后进行下一轮克隆化；挑选生长活力强的ev-d68单一毒株作为溶瘤病毒候选毒株。1.2基于感染性克隆与反向遗传学技术获得肠道病毒ev-d68及其修饰形式的拯救毒株本实施例以野生型ev-d68(seqidno:1)为示例，展示如何通过反向遗传学技术获得用于本发明的ev-d68及其修饰形式，具体方法如下所述。(1)病毒感染性克隆构建：野生型肠道病毒ev-d68(命名为ev-d68-wt)的cdna序列见seqidno:1，其基因组rna序列为seqidno:12；或者在野生型肠道病毒ev-d68的cdna(seqidno:1)的基础上做基因插入或替换改造，包括：修饰形式1：将野生型ev-d68的内部核糖体进入位点序列替换为人鼻病毒2的内部核糖体进入位点序列(其dna序列见seqidno:20)，从而获得重组病毒(命名为ev-d68-hrv2)的cdna(seqidno:8)，其基因组rna序列为seqidno:13；修饰形式2：将mir-133靶序列(其dna序列见seqidno:17)和mir-206靶序列(其dna序列见seqidno:18)的串联序列(其dna序列见seqidno:19)插入到野生型ev-d68的cdna(seqidno:1)的3’非翻译区7293-7294bp之间，从而获得重组病毒(命名为ev-d68-mir133&206t)的cdna(seqidno:9)，其基因组rna序列为seqidno:14；修饰形式3：将人粒细胞-巨噬细胞集落刺激因子(gm-csf)基因(seqidno:6)插入到野生型ev-d68的cdna(seqidno:1)的vp1与2a基因之间，从而获得重组病毒(命名为ev-d68-gm-csf)的cdna(seqidno:10)，其基因组rna序列为seqidno:15；修饰形式4：将编码抗人程序性死亡受体1单链抗体(anti-pd-1scfv)的序列(seqidno:7)插入到野生型ev-d68的cdna(seqidno:1)的vp1与2a基因之间，从而获得该重组病毒(命名为ev-d68-anti-pd-1)的cdna(seqidno:11)，其基因组rna序列为seqidno:16。然后将上述五种溶瘤病毒的cdna序列(seqidno:1,8-11)送到基因合成公司(上海生工生物工程股份有限公司)进行全基因合成，并连接进入psva质粒(hou等人，virusres2015,205:41-44；yang等人，virusres2015,210:165-168)，获得肠道病毒ev-d68或其修饰形式(即，ev-d68-wt、ev-d68-hrv2、ev-d68-mir133&206t、ev-d68-gm-csf和ev-d68-anti-pd-1)的感染性克隆质粒。(2)质粒小提试剂盒和ecoli.dh5α感受态购自北京天根生化科技有限公司；hela细胞(number:ccl-2tm)和人横纹肌肉瘤细胞(rd细胞；number:ccl-136tm)由中国，厦门大学，国家传染病诊断试剂与疫苗工程技术研究中心保存，分别用dmem和mem培养基、加10％胎牛血清以及谷氨酰胺、青霉素和链霉素培养；转染试剂lipofactamine2000和opti-mem购自thermofisherscientific公司。(3)将分别包含上述五种溶瘤病毒cdna序列的感染性克隆质粒转化进入ecoli.dh5α感受态，待克隆长出后直接挑取单克隆菌株摇菌后用质粒小提试剂盒提取质粒，随后送公司(上海生工生物工程股份有限公司)进行测序分析；(4)将测序正确的感染性克隆质粒与辅助质粒par3126共转染细胞拯救病毒(hou等人，virusres2015,205:41-44；yang等人，virusres2015,210:165-168)。首先转染hela细胞，具体操作方法参见转染试剂说明书；随后在显微镜下观察，当hela细胞出现cpe时收获细胞及培养上清，并接种至rd细胞进行传代与培养，获得的拯救病毒可作为溶瘤病毒候选毒株。实施例2：ev-d68及其修饰形式的体外抗肿瘤实验2.1所使用的病毒与细胞系(1)病毒：本实施例使用实施例1所提供的ev-d68-wt(seqidno:12)、ev-d68-hrv2(seqidno:13)、ev-d68-mir133&206t(seqidno:14)、ev-d68-gm-csf(seqidno:15)和ev-d68-anti-pd-1(seqidno:16)。(2)细胞系：人横纹肌肉瘤细胞rd(number:ccl-136tm)；人宫颈癌细胞系hela(number:ccl-2tm)、siha(number:htb-35tm)、caski(number:crl-1550tm)和c-33a(number:htb-31tm)；人卵巢癌细胞系skov-3/tr(skov-3耐药株)、skov-3(number:htb-77tm)和caov3(number:htb-75tm)；人子宫内膜癌细胞系hec-1-a(number:htb-112tm)、hec-1-b(number:htb-113tm)和ishikawa(ecaccno.99040201)；人肺癌细胞系spc-a-1(cctcc保藏编号:gdc050)、nci-h1299(number:crl-5803tm)、nci-h1417(number:crl-5869tm)、nci-h1703(number:crl-5889tm)、nci-h1975(number:crl-5908tm)、a549(number:ccl-185tm)、nci-h661(number:htb-183tm)、ebc-1(thermofisherscientific,catalog#:11875101)和dms114(number:crl-2066tm)；人肝癌细胞系mhcc97h(购自复旦大学肝癌研究所)、c3a(number:crl-10741tm)、hep3b(number:hb-8064tm)、hepg2(number:hb-8065tm)、smmc7721(购自中国医学科学院基础医学研究所基础医学细胞中心，编号:3111c0001ccc000087)、bel7402(cctcc保藏编号:gdc035)、bel7404(购自中国科学院上海生命科学研究院细胞资源中心，编号:3131c0001000700064)、huh7(cctcc保藏编号:gdc134)、plc/prf/5(number:crl-8024tm)和sk-hep-1(number:htb-52tm)；人肾癌细胞系a-498(number:htb-44tm)、786-o(number:crl-1932tm)和caki-1(number:htb-46tm)；人神经母细胞瘤细胞系sh-sy5y(number:crl-2266tm)和sk-n-be(2)(number:crl-2271tm)；人神经胶质瘤细胞系u87-mg(number:htb-14tm)和u118-mg(number:htb-15tm)；人乳腺癌细胞系mcf-7(number:htb-22tm)、bcap37(cctcc保藏编号:gdc206)、bt-474(number:htb-20tm)、mda-mb-231(number:crm-htb-26tm)和mda-mb-453(number:htb-131tm)；人黑色素瘤细胞系a-375(number:crl-1619tm)、sk-mel-1(number:htb-67tm)和mewo(number:htb-65tm)；人前列腺癌细胞系pc-3(number:crl-1435tm)、lncap(number:crl-1740tm)和du145(number:htb-81tm)；人膀胱癌细胞系j82(number:htb-1tm)和5637(number:htb-9tm)；人胰腺癌细胞系capan-2(number:htb-80tm)、hpaf-2(number:crl-1997tm)和panc-1(number:crl-1469tm)；人胃癌细胞系ags(number:crl-1739tm)、sgc7901(cctcc保藏编号:gdc150)、bgc823(cctcc保藏编号:gdc151)和nci-n87(number:crl-5822tm)；人结直肠癌细胞系dld-1(number:ccl-221tm)、sw1116(number:ccl-233tm)、sw480(number:ccl-228tm)、hct-116(number:ccl-247tm)和ht-29(number:htb-38tm)；人食管癌细胞系te-1(购自中国科学院上海生命科学研究院细胞资源中心，编号:3131c0001000700089)；人甲状腺癌sw-579(number:htb-107tm)和tt(number:crl-1803tm)；人喉癌hep-2(number:ccl-23tm)；成骨肉瘤143b(number:crl-8303tm)和u2os(number:htb-96tm)；人淋巴瘤和白血病细胞系k562(number:ccl-243tm)、u937(number:crl-1593.2tm)、thp-1(number:tib-202tm)、raji(number:ccl-86tm)、daudi(number:ccl-213tm)、jurkat(number:tib-152tm)和mt-4(来源于美国国立卫生研究院)；人正常细胞系包括：人胚肺成纤维细胞系mrc-5(number:ccl-171tm)、人胚肾细胞系hek-293(number:crl-1573tm)、人包皮成纤维细胞系hff-1(number:scrc-1041tm)、人皮肤角化细胞系hacat(cctcc保藏编号:gdc106)、人前列腺基质细胞系wpmy-1(number:crl-2854tm)、人脐静脉内皮细胞系huvec(thermofisherscientific,catalog#:c01510c)以及分化后的人肝祖细胞系heparg(具原代肝细胞特征；thermofisherscientific,catalog#:hprgc10)。以上细胞均由中国，厦门大学，国家传染病诊断试剂与疫苗工程技术研究中心保存。heparg细胞在wme培养基(添加1.5％dmso)中培养，ags和tt使用f-12k培养基，sw-579使用l-15培养基，sh-sy5y和sk-n-be(2)使用dmem:f12(1:1)培养基，rd、c-33a、ebc-1、j82、sk-hep-1、sk-mel-1和du145使用mem培养基，k562、u937、thp-1、raji、daudi、jurkat、mt-4、5637、786-o、te-1、caski、nci-h1417、nci-h1703、nci-h1975、nci-h661、sgc7901、bgc823、dld-1、sw1116、hep-2和lncap使用rpmi-1640培养基，其他细胞都使用dmem培养基，这些培养基均需添加10％的胎牛血清、谷氨酰胺与青霉素-链霉素双抗。以上所有细胞在37℃，5％co2的标准条件下培养。2.2病毒的培养将rd细胞均匀铺于10厘米细胞培养板上，培养条件为含有10％胎牛血清以及谷氨酰胺、青霉素和链霉素的mem培养基，37℃，5％co2，饱和湿度；待细胞汇合度达到90％以上时，将细胞培养基更换为无血清的mem培养基，每板接种107tcid50的ev-d68-wt、ev-d68-hrv2、ev-d68-mir133&206t、ev-d68-gm-csf或ev-d68-anti-pd-1，培养环境更换为33℃，5％co2，饱和湿度；24小时后，ev-d68或其修饰形式在rd细胞内增殖并引起细胞出现cpe；当90％以上细胞出现收缩变圆、颗粒感增加以及脱落裂解时，收获细胞及其培养液上清；经3次反复冻融后，收集培养上清进行离心除去细胞碎片，离心条件为4000rpm，10min，4℃；最后，用0.22μm的一次性滤器(millipore公司)过滤上清，除尽细胞碎片等杂质。2.3病毒滴度的测定将rd细胞铺于96孔板当中，细胞密度为104个/孔；待细胞贴壁之后，对实施例2.2中获得的病毒液用无血清mem培养基进行10倍起始的10倍梯度稀释。加50μl稀释后的病毒到细胞孔当中，7天后观察并标记出现cpe的细胞孔，利用karber法进行计算，计算公式为lgtcid50＝l-d(s-0.5)，l：最高稀释度的对数，d：稀释度对数之间的差，s：阳性孔比率总和。此时计算出来的tcid50单位为tcid50/50μl，需换算成tcid50/ml。2.4病毒的体外抗肿瘤实验将人肿瘤细胞与正常细胞按照104个/孔接种至96孔板；待细胞贴壁，每孔更换为无血清的对应细胞培养基，并且分别接种moi为0.1、1、10和100的病毒；随后，每天在显微镜下观察细胞是否产生cpe。其中，未感染病毒的人脐静脉内皮细胞系huvec、人食管癌细胞系te-1、人甲状腺癌细胞系sw-579和tt(阴性对照组，mock)，以及使用moi＝10ev-d68-wt处理72小时后的上述细胞的显微镜照片如图1所示。结果显示，在感染复数(moi)为10感染72小时后，病毒感染组的肿瘤细胞数量明显减少，且明显皱缩、破裂等现象；而经病毒感染的非肿瘤细胞与mock组的非肿瘤细胞相比，其细胞形态几乎无变化。上述结果表明，ev-d68对人肿瘤细胞系te-1、sw-579和tt均有显著的溶瘤作用，但对非肿瘤细胞huvec无任何影响。在病毒感染培养72小时后使用cellcountingkit-8(cck-8试剂盒；上海碧云天生物技术有限公司)和结晶紫染色的方法(仅用于贴壁细胞)检测细胞存活率，具体方法如下：(1)cck8法检测细胞存活率在96孔细胞培养板中，贴壁细胞直接弃掉原培养基，悬浮细胞经离心后小心弃掉原培养基，随后更换为每孔100μl的新鲜无血清培养基；在接种细胞的孔中每孔加入10μlcck-8溶液，同时在空白培养液中也加入等量的cck-8溶液作为阴性对照；在细胞培养箱内37℃孵育0.5-3小时，在0.5、1、2、3小时分别利用酶标仪在吸光度为450nm进行一次检测，选取吸光度范围比较适宜的时间点作为细胞存活率的参考。ev-d68-wt对各细胞的cck-8检测结果参见表2，其中，“-”表示病毒处理后的细胞存活率与mock组相比无显著性差异；“+”表示病毒处理后的细胞数量减少但存活率仍大于50％，且与mock组相比具有显著性差异；“++”表示病毒处理后的细胞存活率小于50％，且与mock组相比具有显著性差异。细胞存活率的计算方法为：(2)结晶紫染色检测细胞存活率(仅用于贴壁细胞)在病毒感染细胞3天后，弃去96孔细胞培养板中的培养上清液，每孔加入100μl甲醇，避光固定15min；称量结晶紫粉末(上海生工生物工程股份有限公司)，配成2％(w/v)的结晶紫甲醇溶液于4℃保存；取适量的2％的结晶紫甲醇溶液用pbs溶液配成0.2％的结晶紫工作溶液；固定15min后，弃去96孔细胞培养板中甲醇固定液，加入100μl的结晶紫工作溶液染色30min；弃去结晶紫染液，用pbs溶液洗3～5次，直至多余染液洗净，空气干燥；利用immunspot@s5uvanalyzer(cellulartechnologylimited,usa)进行拍照，记录实验结果。其中，人肝癌细胞系hepg2、smmc7721、bel7404、bel7402和huh7；人宫颈癌细胞系hela和caski；人肺癌细胞系nci-h1299和a549；人包皮成纤维细胞系hff-1、人胚肾细胞系hek-293和分化后的人肝祖细胞系heparg的对照组(mock)和实验组(使用moi分别为0.1，1和10的ev-d68-wt感染72小时)的结晶紫染色结果如图2所示。结果显示，在moi为10、1和0.1感染72小时后，与未加入病毒的对照组(mock)相比，实验组中的各肿瘤细胞的细胞数量明显减少；但非肿瘤细胞的细胞数量变化不明显。上述结果表明ev-d68-wt对人肿瘤细胞系hepg2、smmc7721、bel7404、bel7402、huh7、hela、caski、nci-h1299和a549均有显著的溶瘤作用，但对非肿瘤细胞系hff-1,、hek-293和分化后的heparg影响很小。表2：野生肠道病毒ev-d68体外抗肿瘤实验结果注：“-”表示病毒处理后的细胞存活率与mock组相比无显著性差异；“+”表示病毒处理后的细胞数量减少但存活率仍大于50％，且与mock组相比具有显著性差异；“++”表示病毒处理后的细胞存活率小于50％，且与mock组相比具有显著性差异。由表2可知，野生肠道病毒ev-d68对所有检测的肿瘤细胞都有杀伤作用，因此具有广谱的抗肿瘤活性；特别的，该病毒对肝癌细胞系、神经胶质瘤细胞系、前列腺癌细胞系、白血病和淋巴瘤细胞系杀伤显著。另一方面，该病毒除了对人胚肺成纤维细胞mrc-5在较高moi时具有明显的毒性，对其他测试的非肿瘤细胞系毒性很弱或没有毒性。另外，ev-d68-hrv2、ev-d68-mir133&206t、ev-d68-gm-csf和ev-d68-anti-pd-1的体外抗肿瘤实验结果显示，上述四种经修饰的ev-d68均保留了母本毒株野生肠道病毒ev-d68对所有检测肿瘤细胞的广谱杀伤作用、以及基本保留了对所检测人肝癌细胞系、前列腺癌细胞系、白血病和淋巴瘤细胞系肿瘤细胞的显著杀伤作用，仅ev-d68-hrv2的溶瘤活性相对有所减弱。其中，这四种经修饰的ev-d68对宫颈癌细胞系hela、神经胶质瘤细胞系u118-mg、肝癌细胞系huh7、前列腺癌细胞系pc-3、淋巴瘤细胞系raji的溶瘤效果的cck-8检测结果如表3所示。表3：ev-d68-hrv2、ev-d68-mir133&206t、ev-d68-gm-csf和ev-d68-anti-pd-1的体外抗肿瘤实验结果注：“-”表示病毒处理后的细胞存活率与mock组相比无显著性差异；“+”表示病毒处理后的细胞数量减少但存活率仍大于50％，且与mock组相比具有显著性差异；“++”表示病毒处理后的细胞存活率小于50％，且与mock组相比具有显著性差异。2.5ev-d68的连续适应性传代在本实施例中，通过将ev-d68在某一种肿瘤细胞内连续适应性传代以获得对该肿瘤细胞杀伤活性增强毒株。将野生肠道病毒ev-d68在其溶瘤作用效果不是非常显著的人宫颈癌细胞系siha、人卵巢癌细胞系skov-3、人肝癌细胞系sk-hep-1、人胰腺癌细胞系capan-2、人胃癌细胞系ags或人结直肠癌细胞系hct-116中分别进行连续适应性传代，具体方法如下：将以上肿瘤细胞其中一种均匀铺于10厘米细胞培养板上，培养条件为含有10％胎牛血清以及谷氨酰胺、青霉素和链霉素的对应细胞培养基，37℃，5％co2，饱和湿度；待细胞汇合度达到90％以上时，将细胞培养基更换为无血清的细胞培养基，每板接种107tcid50的ev-d68病毒，培养环境更换为33℃，5％co2，饱和湿度；待ev-d68在肿瘤细胞内增殖并引起细胞出现cpe时(感染最多3天后)，收获细胞及其培养液上清；经三次冻融后，4℃，4000rpm离心10min；取离心上清加入细胞汇合度达到90％以上的新的肿瘤细胞中，完成一次病毒传代。如此反复传代10次以上，每传代一次取出部分病毒液在rd细胞中进行病毒滴度检测，具体方法参照实施例2.3。通常病毒复制能力会随代次增加呈增强趋势，直至可在该肿瘤细胞中达到较高的感染性滴度且病毒复制稳定，即获得该肿瘤细胞的ev-d68适应毒株。随后，通过实施例2.4中所述的体外抗肿瘤实验的操作方法，将人肿瘤细胞siha、skov-3、sk-hep-1、capan-2、ags或hct-116按照104个/孔接种至96孔板；待细胞贴壁，每孔更换为无血清的对应细胞培养基，37℃孵育30min，再分别接种moi为0.1、1、10和100的每种细胞相对应的连续传代ev-d68适应毒株(病毒滴度在rd细胞上检测)；随后每天在显微镜下观察细胞是否产生cpe，并在病毒感染培养72小时后使用cck-8法检测细胞存活率。结果如表4所示，野生肠道病毒ev-d68经过在其溶瘤作用效果不好的某一种肿瘤细胞中连续传代后，其对该肿瘤细胞的杀伤活性明显增强，表明可通过上述连续传代的方法获得对该肿瘤细胞溶瘤作用效果增强的ev-d68适应毒株。表4：通过在某肿瘤细胞内连续适应性传代后的ev-d68对该肿瘤细胞的体外杀伤实验结果注：“-”表示病毒处理后的细胞存活率与mock组相比无显著性差异；“+”表示病毒处理后的细胞数量减少但存活率仍大于50％，且与mock组相比具有显著性差异；“++”表示病毒处理后的细胞存活率小于50％，且与mock组相比具有显著性差异。2.6ev-d68基因组rna的溶瘤效果评价在本实施例中，通过将纯化的ev-d68基因组rna转染某一种肿瘤细胞可产出大量具有感染性的ev-d68活病毒并对肿瘤细胞造成杀伤。首先通过体外转录的方法以获得病毒基因组rna，该方法可参见例如hadacem,kellyejandrussellsj.molther，2011,19(6):1041–1047。具体而言，将实施例1中所获得的野生型ev-d68的感染性克隆质粒线性化，以该线性化质粒为模板，使用试剂盒megascripttmt7transcriptionkit(thermofisherscientific,am1333)体外转录并大量生产病毒rna，再使用megacleartmtranscriptionclean-upkit(thermofisherscientific,am1908)纯化获得的病毒rna待用，4份平行样品的rna电泳图谱如图3所示。随后，通过实施例2.4中所述的体外抗肿瘤实验的操作方法，将人宫颈癌肿瘤细胞系hela按照105个/孔接种至24孔板；待细胞贴壁，每孔更换为无血清的对应细胞培养基，37℃孵育30min，再使用转染试剂2000(thermofisherscientific,11668019)将纯化好的病毒rna按照1μg每孔转染hela细胞，阴性对照组转染无关rna核酸分子，随后每天在显微镜下观察细胞是否产生cpe。结果发现，转染ev-d68基因组rna的hela细胞在转染后8小时左右开始出现cpe，随后细胞病变逐渐加剧，48小时后使用cck8法检测存活率，hela细胞已经几乎全部死亡裂解，hela细胞感染0、24和48小时后的显微镜照片如图4所示；培养上清接种到新的hela细胞中又可很快产生cpe。该结果表明，直接施用ev-d68的核酸亦具有良好的杀伤活性，可用于治疗肿瘤。实施例3：肠道病毒ev-d68及其修饰形式的体内抗肿瘤实验3.1病毒、细胞系与实验动物(1)病毒：本实施例中使用实施例1所提供的ev-d68-wt(seqidno:12)、ev-d68-hrv2(seqidno:13)、ev-d68-mir133&206t(seqidno:14)、ev-d68-gm-csf(seqidno:15)和ev-d68-anti-pd-1(seqidno:16)。病毒的培养与病毒滴度测定方法分别参见实施例2.2和2.3。(2)细胞系：人宫颈癌细胞系hela(number:ccl-2tm)、神经胶质瘤细胞系u118-mg(number:htb-15tm)、淋巴瘤细胞系raji(number:ccl-86tm)。除raji使用rpmi-1640培养基培养，其他hela和u118-mg均使用dmem培养基培养，这些培养基均需添加10％的胎牛血清、谷氨酰胺与青霉素-链霉素双抗。以上所有细胞在37℃，5％co2的标准条件下培养。(3)实验动物：6-8周龄的雌性c.b17scid小鼠来源于上海斯莱克实验动物有限责任公司；根据厦门大学实验动物中心与伦理委员会所批准的方案，将该小鼠在spf条件下饲养。3.2病毒的体内抗肿瘤实验将用于scid小鼠皮下成瘤的肿瘤细胞用0.01％胰蛋白酶消化后，再使用含10％的胎牛血清的细胞培养基重悬成单细胞悬液；计数悬液的细胞密度，1000g，3min离心沉淀细胞，再用适量体积的pbs重悬细胞，使达到约106-107个细胞/100μlpbs；按照106-107个细胞/100μlpbs/点在scid小鼠背部皮下用注射器接种肿瘤细胞，待14-21天左右、肿瘤细胞在scid小鼠皮下形成大约100mm3的瘤块的时候，将荷瘤scid小鼠随机分为实验组(施用ev-d68-wt、ev-d68-hrv2、ev-d68-mir133&206t、ev-d68-gm-csf或ev-d68-anti-pd-1)与阴性对照组，每组4只(n＝4)，用106tcid50/100μl无血清培养基/每瘤块的溶瘤病毒(ev-d68-wt、ev-d68-hrv2、ev-d68-mir133&206t、ev-d68-gm-csf或ev-d68-anti-pd-1)或等量无血清培养基瘤内注射处理，每两天注射1次，共处理5次。每两天用游标卡尺测量并记录肿瘤大小变化，肿瘤大小的计算方法为：肿瘤大小(mm3)＝肿瘤长度数值×(肿瘤宽度数值)2/2。ev-d68-wt对上述三种肿瘤的治疗结果分别如图5a-5c所示。结果显示，攻毒ev-d68-wt后，所检测的hela(a)、u118-mg(b)和raji(c)三种肿瘤生长均出现逐渐变慢和停滞，甚至裂解消失；相比之下，阴性组(ctrl)肿瘤则保持正常生长，其肿瘤大小显著大于实验组。表5显示了ev-d68-wt、ev-d68-hrv2、ev-d68-mir133&206t、ev-d68-gm-csf和ev-d68-anti-pd-1分别治疗raji肿瘤模型10天后的结果。结果显示，与未经溶瘤病毒治疗的阴性对照组相比，ev-d68-wt、ev-d68-hrv2、ev-d68-mir133&206t、ev-d68-gm-csf和ev-d68-anti-pd-1治疗后的肿瘤体积显著减小，其中ev-d68-wt、ev-d68-mir133&206t、ev-d68-gm-csf和ev-d68-anti-pd-1这4种溶瘤病毒处理后肿瘤体积减小幅度类似。上述结果表明ev-d68-wt、ev-d68-hrv2、ev-d68-mir133&206t、ev-d68-gm-csf和ev-d68-anti-pd-1均展现出显著有利的体内抗肿瘤活性。表5：ev-d68-wt、ev-d68-hrv2、ev-d68-mir133&206t、ev-d68-gm-csf和ev-d68-anti-pd-1对人淋巴瘤细胞系raji的体内抗肿瘤实验结果溶瘤病毒治疗10天后对raji的体内溶瘤效果ev-d68-wt++ev-d68-hrv2+ev-d68-mir133&206t++ev-d68-gm-csf++ev-d68-anti-pd-1++注：“+”表示治疗后肿瘤体积缩小但仍大于阴性对照组的50％，且与阴性对照组相比具有显著性差异；“++”表示治疗后肿瘤体积缩小至小于阴性对照组的50％，且与阴性对照组相比具有显著性差异。实施例4：溶瘤病毒的安全性评价4.1所用的病毒与实验动物(1)病毒：本实施例使用由实施例1所提供的ev-d68-wt(seqidno:12)。病毒的培养与病毒滴度测定方法分别参照实施例2.2和2.3。(2)实验动物：balb/c孕鼠来源于上海斯莱克实验动物有限责任公司；根据厦门大学实验动物中心与伦理委员会所批准的方案，将该小鼠在清洁级条件下饲养，随后选取balb/c孕鼠生产的1日、2日、3日、7日和14日小鼠用于ev-d68的体内毒力评估。4.2病毒在小鼠体内的安全性评价(1)选取balb/c1日龄乳鼠进行ev-d68-wt的腹腔攻毒，攻毒滴度剂量为103、104、105、106或107tcid50/只，随后每天对不同剂量攻毒组balb/c小鼠存活率和健康分数进行记录，健康分数的评定标准为：5分，濒死或死亡；4分，肢体重症瘫痪；3分，肢体无力或轻度畸形；2分，消瘦；1分，萎靡、竖毛、颤抖；0分，健康。结果如图6a所示。在攻毒后的20天内，极高剂量107tcid50组小鼠1周内全部发病与死掉；高剂量106tcid50组最终80％小鼠存活下来，仅少量小鼠出现发病与死亡；除此之外，其他剂量攻毒组小鼠均未发病与死亡。(2)选取出生后1日、2日、3日、7日和14日的balb/c小鼠分别进行极高剂量107tcid50/只的ev-d68-wt腹腔注射，随后每天对不同日龄组balb/c小鼠的存活率以及健康分数进行记录，健康分数的评定标准同上。结果如图6b所示。在攻毒后的20天内，1日龄小鼠1周内全部发病死亡；2日龄小鼠对ev-d68毒性有所抵抗，最终有70％的存活率，但发病率偏高且症状较重；3日龄小鼠已经对ev-d68不敏感，最终有90％的存活率，发病低且症状轻；4日龄以上小鼠已经对高剂量的ev-d68完全耐受，无任何发病与死亡症状发生。上述结果表明，ev-d68-wt对小鼠的毒性较弱，仅在使用极高剂量的107tcid50/只时对1-3日龄balb/c小鼠致死，而对4日龄及以上小鼠无任何影响，具有良好的体内安全性。尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。序列表<110>厦门大学北京万泰生物药业股份有限公司<120>一种用于治疗肿瘤的病毒<130>idc170023<160>20<170>patentinversi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gtcccccggcgagagggctacactgagctgcagggcctcc540aagggcgtgtccacctccggctactcctacctgcactggtaccagcagaagcctggacag600gctcccaggctgctgatctacctggcctcctacctggagtccggcgtgcctgctaggttt660tccggcagcggcagcggcaccgatttcaccctgaccatctcctccctggagcccgaggac720ttcgccgtgtactactgccagcactccagggatctgcctctgaccttcggcggcggcacc780aaggtggagatcaag795<210>8<211>7306<212>dna<213>人工序列<220><223>ev-d68-hrv2的cdna序列<400>8taatacgactcactataggttaaaacagccttggggttgttcccactccaagggcccacg60tggcggctagtactctggtacttcggtacctttgtacgcctgttttatctcccttcccaa120tgtaacttagaagaacttagaagtttttcacaaagaccaatagccggtaatcagccagat180tactgaaggtcaagcacttctgtttccccggtcaatgttgatatgctccaacagggcaaa240aacaactgcgatcgttaaccgcaaagcgcctacgcaaagcttagtagcatctttgaaatc300gtttggctggtcgatccgccatttcccctggtagacctggcagatgaggctagaaatacc360ccactggcgacagtgttctagcctgcgtggctgcctgcacaccctatgggtgtgaagcca420aacaatggacaaggtgtgaagagccccgtgtgctcgctttgagtcctccggcccctgaat480gtggctaaccttaaccctgcagctagagcacgtaacccaatgtgtatctagtcgtaatga540gcaattgcgggatgggaccaactactttgggtgtccgtgtttcactttttcctttatatt600tgcttatggtgacaatatatacaatatatatattggcaccatgggagctcaggttactag660acaacaaactggcactcatgaaaatgccaacattgccacaaatggatctcatatcacata720caatcagataaacttttacaaggatagctatgcggcttcagccagcaagcaggatttttc780acaggacccatcaaaattcactgaaccagtagtggaaggtttaaaagcaggggcgccagt840tttgaaatctcctagtgctgaggcatgtggctacagtgatagagtattacagctcaaatt900aggaaattcagctattgtcacccaggaagcagcgaactactgctgcgcttatggtgaatg960gcccaattacttaccagaccatgaagcagtagccattgataaacctacacaaccagaaac1020tgctacagatagattctacactttgaaatcagtcaaatgggaaactggaagcacaggatg1080gtggtggaaactacccgatgcactgaataatataggcatgtttggacagaatgtgcagca1140tcactacctatatagatctggtttcttgattcatgtgcagtgtaatgccacaaaattcca1200tcaaggtgccttattagtggtagcaattccagaacatcagaggggagcgcacaacaccaa1260cactagcccagggtttgatgatataatgaaaggtgaagaaggagggaccttcaatcatcc1320atatgtccttgatgatggaacatcattggcttgtgcgacgatatttccacatcagtggat1380aaatctgagaaccaacaattcagcaacaattgttcttccctggatgaatgctgctccaat1440ggatttcccacttagacataatcagtggacgctagcaataataccagtggtgccattagg1500tacgcgtacaacatcaagtatggtcccaataacagtttcaatcgctccaatgtgttgtga1560gtttaatggacttagacacgccattactcaaggtgtcccaacataccttttaccaggctc1620gggacaattcctaacaactgatgatcatagctctgcaccagctctcccgtgtttcaaccc1680aactccagaaatgcatatcccagggcaggtccgtaacatgctagaagtggtccaagtgga1740atcaatgatggagattaataacacagaaagtgcagttggcatggagcgtcttaaggttga1800tatatcagcattgacagatgtcgatcaattgttattcaacattccactggacatacagtt1860ggatgggccacttagaaacactttggtaggaaacatatctagatattacactcattggtc1920tggatccctagaaatgacgtttatgttttgtggcagcttcatggcaacgggaaaattaat1980cctgtgctatactcctccaggtggatcatgcccgacaaccagagagaccgccatgttagg2040tacacatattgtttgggattttggattacaatctagtgtaaccctgataataccttggat2100tagtggatcccactacaggatgtttaataatgatgctaagtcaactaatgccaacgttgg2160ctatgtcacttgttttatgcagaccaatctgatagtccccagtgaatcctctgacacgtg2220ttccttgatagggttcatagcagcaaaagatgatttctccctcagattaatgagagacag2280ccctgacattggacaactagaccatttacatgcagcagaggcagcctaccagatcgagag2340catcatcaaaacagcgaccgacactgtgaaaagtgagattaatgctgaacttggtgtggt2400ccctagcttaaatgcagttgaaacaggtgcaacttctaacactgaaccagaagaagccat2460acaaactcgcacagtgataaatcagcacggtgtatccgagactctagtggagaattttct2520cagtagagcagctttggtatcaaagagaagttttgaatacaaagatcatacttcgtctgc2580agcacaagcagacaagaactttttcaaatggacaattaacaccagatcctttgtacagtt2640aagaagaaaattagaattattcacataccttagatttgatgctgagatcactatactcac2700aactgtagcagtgaatggtagtggtaataatacatacgtgggtcttcctgacttgacact2760ccaagcaatgtttgtacccactggtgctcttaccccagaaaaacaggactcattccactg2820gcagtcaggcagtaatgctagtgtattctttaaaatctccgaccccccagccagaataac2880cataccttttatgtgcattaactcagcatactcagttttttatgatggctttgccggatt2940tgagaaaaacggtctgtatggaataaatccagctgacactattggtaacttatgtgttag3000aatagtgaatgaacaccaaccagttggtttcacagtgaccgttagggtttacatgaagcc3060taaacacataaaagcatgggcaccacgaccaccacgaactttgccatatatgagtattgc3120aaatgcaaattacaaaggtaaagaaagagcaccaaatgcgctcaatgctataattggcaa3180tagagacagtgtcaaaaccatgcctcataatatagtgaacactggtccaggcttcggagg3240agtttttgtagggtctttcaaaataatcaactatcacttggccactacagaagagagaca3300gtcagctatctatgtggattggcaatcagacgtcttggttacccccattgctgctcatgg3360aaggcaccaaatagcaagatgcaagtgcaacacaggggtttactattgtaggcacaaaaa3420cagaagttacccgatttgctttgaaggcccagggattcaatggattgaacaaaatgaata3480ttacccagcaaggtaccagaccaatgtactattggcagttggtcctgcggaagcaggaga3540ttgcggtggtttactagtttgtccacatggggtaatcggtcttcttacagcaggaggggg3600tggaattgtagctttcactgatatcaggaatttgctatggttagatactgatgctatgga3660acaaggcattactgattatattcaaaatcttggtaatgcctttggagcaggatttacaga3720aacaatctctaataaagccaaggaagtgcaagatatgctaattggagagagttcactatt3780agaaaaattgttaaaagctctaatcaaaatcatatcagcattagtaattgtaatcagaaa3840ctcagaagatttagtcacagtcacagccacactagcattgttgggatgccatgattcacc3900atggagctacttgaaacagaaggtatgttcatacttaggtattccttatgtacctagaca3960gggtgaatcgtggcttaagaaattcacagaggcatgcaatgctcttagaggtctggattg4020gctatcgcaaaagatagataaattcatcaactggcttaaaaccaaaatattaccagaagc4080tagggagaaatatgaatttgtgcaaaggctcaaacagttaccggtgatagaaaaccaagt4140tagtacaatcgagcatagctgcccaacaacagaacaacaacaggccttattcaacaacgt4200ccaatactattcacactactgtagaaagtacgcaccactttacgcagtggaagcaaagag4260ggtagtagctcttgaaaagaaaataaacaactacatccagttcaagtccaaatctcgcat4320tgaaccggtttgtttaataatacatggctc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