近日，国际著名杂志Virology Journal在线刊登了中国科学院上海巴斯德研究所关于构建手足口病相关的柯萨奇病毒A16型（coxsackievirus A16，CVA16）感染性克隆的最新研究成果。
手足口病是5岁以下儿童中发生的常见传染病，仅2010年和2011年，全国共有3，394，375例感染，其中死亡1414例，对于儿童的健康成长造成严重威胁，但目前尚无针对手足口病的预防性疫苗或治疗性药物。
CVA16和人肠道病毒71型(enterovirus 71，EV71)是引起手足口病的主要病原体，因此，了解CVA16和EV71基因组中各个区域的功能，对加强CVA16和EV71的病毒学和免疫学研究，尽快研制出有效的预防和治疗方法，保障中国儿童的生命健康有非常重要的意义。然而，EV71和CVA16均属于RNA病毒，直接对它们的基因组进行遗传操作非常困难。
上海巴斯德所研究生刘菲、刘庆伟、蔡一村等在黄忠研究员指导下，通过分子生物学手段，提取了CVA16病毒的基因组RNA，并且进行体外反转录操作获得与病毒基因组相对应的cDNA序列，将此序列构建到一个表达载体中，获得携带感染性cDNA克隆的质粒。利用该质粒可以包装出具有感染性的拯救病毒。通过观察细胞病变、蛋白质印迹以及免疫荧光等方法证实，拯救病毒与野生病毒的功能完全一致。同时，测序结果还表明，拯救病毒的基因组具有稳定的遗传性。
利用新构建出的感染性克隆，科研人员可以很方便地对RNA病毒的基因组进行体外操作，从而对病毒的结构、功能等多方面进行研究。更长远地来说，CVA16感染性克隆为今后阐明病毒的致病性，开发针对EV71/CVA16的预防性疫苗和治疗性药物等提供了研究基础。
该研究得到中国科学院“百人计划”、生化工程国家重点实验室开放基金及赛诺菲-安万特-中科院上海生命科学研究院优秀青年人才基金的资助。（生物谷Bioon.com）
doi:10.1186/1743-422X-8-534
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Construction and characterization of an infectious clone of coxsackievirus A16
Fei Liu†, Qingwei Liu†, Yicun Cai†, Qibin Leng and Zhong Huang*
Background Coxsackievirus A16 (CVA16) is a member of the Enterovirus genus of the Picornaviridae family and it is a major etiological agent of hand, foot, and mouth disease (HFMD), which is a common illness affecting children. CVA16 possesses a single-stranded positive-sense RNA genome containing approximately 7410 bases. Current understanding of the replication, structure and virulence determinants of CVA16 is very limited, partly due to difficulties in directly manipulating its RNA genome.
Results Two overlapping cDNA fragments were amplified by RT-PCR from the genome of the shzh05-1 strain of CVA16, encompassing the nucleotide regions 1-4392 and 4381-7410, respectively. These two fragments were then joined via a native XbaI site to yield a full-length cDNA. A T7 promoter and poly(A) tail were added to the 5' and 3' ends, respectively, forming a full CVA16 cDNA clone. Transfection of RD cells in vitro with RNA transcribed directly from the cDNA clone allowed the recovery of infectious virus in culture. The CVA16 virus recovered from these cultures was functionally and genetically identical to its parent strain.
Conclusions We report the first construction and characterization of an infectious cDNA clone of CVA16. The availability of this infectious clone will greatly enhance future virological investigations and vaccine development for CVA16
