APPLICATIONS OF INFECTIOUS CDNA TECHNOLOGY TO RNA VIRUS

我们从一种强毒登革热1型病毒（DEN1 WP）和一种已适应在狗肾细胞中生长的DEN1减毒候选活疫苗（DEN1 PDK20）中克隆出全长的“传染性”cDNA克隆。首先，以病毒粒子RNA为模板，合成寡核苷酸引物，采用RT-PCR扩增DEN1 WP的左右端；引物在左端上游引入一个SP6 RNA聚合酶启动子。cDNA产物（左端约3kb，右端约4kb）在质粒pRS424的多联体上定向克隆。然后，用RT-PCR方法从DEN1 WP的中间片段获得5kb的cDNA；这个片段与左右克隆端重叠。在酵母共转染后，将线性化克隆与5kb cDNA进行同源重组，得到含有全长DEN1 WP cDNA的质粒。从大肠杆菌中制备质粒DNA，并进行体外转录。RNA电穿孔进入LLCMK2细胞产生登革热感染。回收的病毒在C6/36细胞上传代。该病毒在生长曲线上表现与亲本den1wp相似。将克隆的DEN1 WP cDNA的核苷酸序列与WP病毒粒子RNA的序列进行比较（通过测序未克隆的RT-PCR产物确定），发现只有5个差异：3个沉默突变和2个错义突变。用PDK-20病毒粒子RNA合成的cDNA取代全长克隆中的WP序列，制备DEN1 PDK20感染性克隆。首先，用一个代表PDK-20右半部分的RT-PCR产物在酵母中通过同源重组取代WP的相应区域。这种嵌合质粒在大肠杆菌中生长，其转录物被证明具有传染性。然后，以嵌合质粒为起点，如上所述引入PDK20的左半部分，鉴定出一个产生感染性转录本的PDK20全长克隆。克隆的DEN1 PDK20 cDNA与PDK20病毒粒子RNA的核苷酸序列比较（通过测序未克隆的RT-PCR产物确定）发现8个差异，包括4个编码变化。这些编码变化在酵母中进一步的同源重组步骤中被修复，从而产生最终的感染性DEN1 PDK20克隆，该克隆与亲本相比只有3个沉默的变化。将转录本电穿孔到Vero细胞中制造病毒，并在C6/36细胞上传代。该病毒具有与其DEN1 PDK20亲本相同的小斑块表型。目前正在研究这些病毒在猴子和狗肾细胞中的生长情况。在未来，我们计划通过在WP和PDK20感染克隆之间制造嵌合体来绘制负责PDK20适应犬细胞生长的突变。此外，在人体临床试验中，PDK20病毒的反应性略强，而PDK26病毒的反应性过弱。我们计划在PDK20克隆中引入更多的减毒突变，以恢复正确的减毒疫苗株，首先我们将使用PDK26中发现的PDK20中没有的突变子集。

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.