CONSTRUCTION OF INFECTIOUS FULL LENGTH CDNA CLONE OF DEN

我们正在从DEN1毒株西太平洋（WP）毒株和候选减毒活疫苗（PDK-20）中制备全长“传染性”DEN1 cdna。DEN1 WP感染性克隆的构建与DEN2感染性克隆的构建方法基本一致。简单地说，通过RT-PCR扩增DEN1西太平洋菌株的左右两端，然后以适当的方向克隆到酵母穿梭载体pRS424的多联体中。该DNA在两个DEN末端之间的多联体中的一个位点上线性化。同时，用RT-PCR方法从DEN1的中间片段合成了一个5kb的cDNA。这个填充片段与pr克隆中包含的左侧和右侧cdna重叠。在酵母共转染的情况下，将stuffer片段与克隆的左侧和右侧cDNA进行双同源重组，得到含有DEN1全长cDNA的环状pRS质粒。通过PCR筛选酵母菌落，将含有DEN1全长cDNA的质粒转染大肠杆菌，制备DNA，并通过限制性内切分析确定其结构。一系列独立克隆的转录本经电穿孔进入细胞后产生典型的登革病毒感染。转录衍生病毒在生长曲线上表现与母体WP病毒相似。测定了其中一个克隆的完整核苷酸序列，WP RNA序列完成度约75%；到目前为止，除了克隆的cDNA中有一个沉默突变外，这两个序列是相同的。用PDK-20减毒病毒的cDNA片段替换FL - DEN1序列的工作已经开始。首先，使用代表PDK-20右半部分的RT-PCR产物替换全长克隆中WP的相同区域。这种嵌合结构的转录本被证明具有传染性。然后，同样用嵌合结构引入PDK-20的左半部分。已确定候选克隆，并正在进行感染性测试。此外，PDK-20 RNA正在测序中。一旦PDK-20的传染性克隆被分离出来，转录本将被制作出来，并由WRAIR的K. Eckels和他的团队在cGMP下引入生产Vero细胞。该候选疫苗随后将由B. Innis及其WRAIR的同事在人体内与亲本PDK-20疫苗病毒进行比较。这将测试病毒群平均序列的传染性克隆在人体内的行为是否与母疫苗病毒相同。拥有一个具有传染性的克隆将允许在PDK-20病毒中引入特定的突变，以实现进一步的衰减。此外，陆军小组也有其他3种登革热血清型的候选疫苗，很可能其中至少有两种没有适当减毒，不能用于人类。因此，在未来，传染性克隆可以从这些其他候选疫苗中衍生出来，并进一步诱变以达到适当的衰减。

We are in the process of making full length "infectious" DEN1 cDNAs from both a virulent DEN1 virus, the Western Pacific (WP) strain, and from a live-attenuated vaccine candidate (PDK-20). The construction of the DEN1 WP infectious clone closely followed the methods used to make the infectious DEN2 clone. Briefly, the left-hand and right-hand ends of DEN1 Western Pacific strain were amplified by RT-PCR and then were cloned in proper orientation into the polylinker of the yeast shuttle vector pRS424. This DNA was linearized at a site in the polylinker between the two DEN ends. Meanwhile, a 5 kb cDNA was made from the middle of DEN1 by RT-PCR. This ~stuffer fragment~ overlaps both the left- and right-hand cDNAs contained in the pRS clone. Double homologous recombination between the stuffer fragment and the cloned left-hand and right-hand cDNAs upon co-transfection of yeast created a circular pRS plasmid containing the full-length DEN1 cDNA. Yeast colonies were screened by PCR, and plasmids containing full-length DEN1 cDNA were transfected into E. coli, DNA was prepared, and the structure confirmed by restriction analysis. Transcripts made from a series of independent clones produced typical dengue virus infections after electroporation into cells. Transcript-derived virus behaved like the parent WP virus in growth curves. The complete nucleotide sequence of one of the clones was determined, and the sequence of the WP RNA is about 75% completed; so far the two sequences are identical save for 1 silent mutation in the cloned cDNA. Work has begun to replace the FL DEN1 sequences with cDNA segments from the attenuated PDK-20 virus. First, an RT-PCR product representing the right half of PDK-20 was used to replace the same region of WP in the full-length clone. Transcripts from this chimeric construct were proven infectious. Then, the chimeric construct was used similarly to introduce the left half of PDK-20. Candidate clones have been identified and are being tested for infectivity. In addition, PDK-20 RNA is being sequenced. Once an infectious clone of PDK-20 is isolated, transcripts will be made and introduced into production Vero cells under cGMP by K. Eckels and his group at WRAIR. This candidate vaccine will then be compared in humans to the parental PDK-20 vaccine virus by B. Innis and colleagues at WRAIR. This will test whether an infectious clone of the average sequence of a virus population will behave the same in people as the parent vaccine virus. Having an infectious clone would allow the introduction ofspecific mutations into the PDK-20 virus in an effort to achieve further attenuation. Also, the Army group also has vaccine candidates for the other 3 dengue serotypes, and it is probable that at least two of these are not properly attenuated for use in humans. Thus, in the future, infectious clones could be derived from these other vaccine candidates and further mutagenized to achieve proper attenuation.