CONSTRUCTION OF INFECTIOUS FULL LENGTH CDNA CLONE OF DEN

我们正在从强毒DEN 1病毒（西太平洋（WP）株）和减毒活疫苗候选物（PDK-20）中制备全长“感染性”DEN 1 cDNA。DEN 1 WP感染性克隆的构建紧密遵循用于制备感染性DEN 2克隆的方法。简言之，通过RT-PCR扩增DEN 1西太平洋菌株的左手端和右手端，然后以适当的方向克隆到酵母穿梭载体pRS 424的多接头中。该DNA在两个DEN末端之间的多接头中的位点处线性化。同时，通过RT-PCR从DEN 1的中部扩增出一个5 kb的cDNA。 该填充片段与pRS克隆中所含的左侧和右侧cDNA重叠。在共转染酵母后，填充片段与克隆的左手和右手cDNA之间的双重同源重组产生了含有全长DEN 1 cDNA的环状pRS质粒。用PCR方法筛选酵母克隆，将含DEN 1全长cDNA的质粒转染E. coli中，制备DNA，并通过限制性分析确认结构。 由一系列独立克隆制成的转录物在电穿孔进入细胞后产生典型的登革病毒感染。 转录衍生的病毒在生长曲线上表现得像亲本WP病毒。测定了其中一个克隆的完整核苷酸序列，WP RNA的序列约完成75%;到目前为止，除了克隆的cDNA中的1个沉默突变外，这两个序列是相同的。 已经开始用来自减毒PDK-20病毒的cDNA片段替换FL DEN 1序列的工作。首先，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。来自该嵌合构建体的转录物被证明具有感染性。然后，类似地使用嵌合构建体以引入PDK-20的左半部分。 候选克隆已经确定，正在进行感染性测试。此外，PDK-20 RNA正在测序中。 一旦分离出PDK-20的感染性克隆，将制备转录物并在cGMP下通过K.埃克尔斯和他的团队在WRAIR。然后由B在人体中将该候选疫苗与亲本PDK-20疫苗病毒进行比较。Innis和他在WRAIR的同事。 这将测试一个病毒群体平均序列的感染性克隆是否会在人体中表现得与亲本疫苗病毒相同。拥有感染性克隆将允许将特定突变引入PDK-20病毒，以实现进一步减毒。 此外，陆军集团也有其他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用于人类。因此，在未来，感染性克隆可以从这些其他候选疫苗中衍生出来，并进一步诱变以实现适当的减毒。

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.