APPLICATIONS OF INFECTIOUS CDNA TECHNOLOGY TO RNA VIRUS

我们从1型登革热病毒（DEN 1 WP）和减毒活DEN 1候选疫苗（DEN 1 PDK 20）中制备了全长“感染性”cDNA克隆，所述候选疫苗已适应于在狗肾细胞中生长。首先，使用病毒体RNA作为模板和合成的寡核苷酸引物，通过RT-PCR扩增DEN 1 WP的左右末端。引物在左手端的上游引入SP 6 RNA聚合酶启动子。将cDNA产物（左端约3 kb，右端约4kb）以适当的方向克隆到质粒pRS 424的多接头中。然后，通过RT-PCR从DEN 1 WP的中部扩增出一个5 kb的cDNA片段，该片段与克隆的左右两端重叠。共转染酵母后，线性化克隆和5 kb cDNA之间的同源重组产生含有全长DEN 1 WP cDNA的质粒。从大肠杆菌中制备质粒DNA，并在体外转录。将RNA电穿孔到LLCMK 2细胞中产生登革热感染。将病毒在C6/36细胞上传代。该病毒在生长曲线上表现得与亲本DEN 1 WP相似。比较克隆的DEN 1 WP cDNA的核苷酸序列与WP病毒体RNA的序列（通过对未克隆的RT-PCR产物进行测序确定），仅发现5个差异：3个沉默突变和2个错义突变。 DEN 1 PDK 20感染性克隆通过用PDK-20病毒体RNA制备的cDNA替换全长克隆中的WP序列来制备。首先，使用代表PDK-20右半部分的RT-PCR产物通过酵母中的同源重组来替换WP的相应区域。该嵌合质粒在E.大肠杆菌，并从它的转录被证明是传染性的。然后，如上所述，以嵌合质粒为起点导入PDK 20的左半部分，鉴定产生感染性转录物的PDK 20全长克隆。克隆的DEN 1 PDK 20 cDNA的核苷酸序列与PDK 20病毒体RNA的核苷酸序列的比较（通过对未克隆的RT-PCR产物测序确定）揭示了8个差异，包括4个编码变化。这些编码变化在酵母中的进一步一系列同源重组步骤中被修复，导致最终的感染性DEN 1 PDK 20克隆，其与其亲本相比仅具有3个沉默变化。将转录物电穿孔到Vero细胞中以制备病毒，所述病毒在C6/36细胞上传代。该病毒具有与其DEN 1 PDK 20亲本相同的小空斑表型。目前正在研究这些病毒在猴和狗肾细胞中的生长。 在未来，我们计划通过在WP和PDK 20感染性克隆之间制造嵌合体，来绘制负责PDK 20适应狗细胞生长的突变。此外，在人体临床试验中，PDK 20病毒的反应原性略高，而PDK 26病毒则过度减毒。 我们计划将进一步的减毒突变引入PDK 20克隆，以恢复正确的减毒疫苗株，首先我们将使用在PDK 26中发现的突变子集，这些突变在PDK 20中没有。

We made full length "infectious" cDNA clones from a virulent dengue type 1 virus (DEN1 WP) and from a live attenuated DEN1 vaccine candidate, which had been adapted to grow in dog kidney cells (DEN1 PDK20). First, the left and right ends of DEN1 WP were amplified by RT-PCR using virion RNA as a template and synthetic oligonucleotide primers. The primers introduce an SP6 RNA polymerase promoter just upstream of the left-hand end. The cDNA products (approximately 3kb at the left end and 4kb at the right end) were cloned in proper orientation in the polylinker of the plasmid pRS424. Then, a 5kb cDNA was made from the middle of DEN1 WP by RT-PCR; this fragment overlaps both the left and right cloned ends. Homologous recombination between the linearized clone and the 5kb cDNA upon cotransfection of yeast creates a plasmid containing the full-length DEN1 WP cDNA. Plasmid DNA was prepared from E coli, and transcribed in vitro. Electroporation of RNA into LLCMK2 cells produced a dengue infection. Recovered virus was passaged on C6/36 cells. This virus behaved like the parent DEN1 WP in growth curves. Comparison of the nucleotide sequence of the cloned DEN1 WP cDNA to the sequence of the WP virion RNA (determined by sequencing uncloned RT-PCR products) revealed only 5 differences: 3 silent and 2 missense mutations. The DEN1 PDK20 infectious clone was made by replacing the WP sequences in the full-length clone with cDNA made from PDK-20 virion RNA. First, an RT-PCR product representing the right half of PDK-20 was used to replace the corresponding region of WP by homologous recombination in yeast. This chimeric plasmid was grown in E. coli, and transcripts from it were proven infectious. Then, the chimeric plasmid was used as a starting point to introduce the left half of PDK20 as above, and a PDK20 full-length clone which made infectious transcripts was identified. Comparison of the nucleotide sequence of the cloned DEN1 PDK20 cDNA with that of PDK20 virion RNA (determined by sequencing uncloned RT-PCR products) revealed 8 differences, including 4 coding changes. These coding changes were repaired in a further series of homologous recombination steps in yeast, resulting in a final infectious DEN1 PDK20 clone which has only 3 silent changes from its parent. Transcripts were electroporated into Vero cells to make virus, which was passaged on C6/36 cells. This virus has the same small plaque phenotype as its DEN1 PDK20 parent. Growth of these viruses in monkey and dog kidney cells is currently being investigated. In the future, we plan to map the mutations responsible for the adaptation of PDK20 to growth in dog cells, by making chimeras between the WP and PDK20 infectious clones. Also, in human clinical trials the PDK20 virus is slightly too reactogenic, yet a PDK26 virus is overattenuated. We plan to introduce further attenuating mutations into the PDK20 clone in an effort to recover a correctly attenuated vaccine strain, and at first we will use subsets of the mutations found in PDK26 which are not in PDK20.