APPLICATIONS OF INFECTIOUS CDNA TECHNOLOGY TO RNA VIRUS

我们从一种强毒的1型登革热病毒(DEN1 WP)和一种已适应在狗肾细胞(DEN1 PDK20)中生长的减毒活疫苗候选株中获得了全长的“有感染性”的cDNA克隆。首先，以病毒RNA为模板，合成寡核苷酸引物，通过RT-PCR扩增出DEN1 WP的左右两端。这些引物在左手端的上游引入了SP6 RNA聚合酶启动子。扩增产物(左端约3kb，右端约4kb)被正确定向克隆到pRS424的多联体中。然后，通过RT-PCR从DEN1 WP的中间片段中扩增出一条5kb的cDNA，该片段与克隆的左右两端重叠。线性化的克隆与5kb的cDNA线性化后的同源重组产生了一个含有全长DEN1 WP cDNA线性化克隆。从大肠杆菌中提取质粒DNA，并在体外转录。将RNA电穿孔到LLCMK2细胞中产生登革热感染。回收的病毒在C6/36细胞上传代。该病毒的生长曲线与亲本DEN1可湿性粉剂相似。将克隆的DEN1 WP基因的核苷酸序列与WP病毒RNA的序列(通过对未克隆的RT-PCR产物测序确定)进行比较，发现只有5个差异：3个沉默突变和2个错义突变。DEN1 PDK20感染性克隆是用PDK-20病毒核糖核酸的cDNA取代全长克隆中的WP序列而得到的。首先，用代表PDK-20右半部分的RT-PCR产物在酵母中用同源重组取代WP的相应区域。这种嵌合质粒在大肠杆菌中生长，其转录本被证明是有感染力的。然后，以该嵌合载体为起点，如上所述，将PDK20的左半部分导入到PDK20中，获得了一个可产生感染性转录本的PDK20全长克隆。将克隆的DEN1 PDK20与PDK20病毒RNA的核苷酸序列进行比较，发现有8个差异，包括4个编码上的变化。这些编码改变在酵母中进一步的一系列同源重组步骤中被修复，导致最终具有感染性的DEN1 PDK20克隆，该克隆与其亲本相比只有3个沉默的改变。将转录物电穿孔到Vero细胞中，制成病毒，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的肾脏细胞中的生长情况。在未来，我们计划通过在WP和PDK20感染性克隆之间制造嵌合体来定位导致PDK20适应狗细胞生长的突变。此外，在人体临床试验中，PDK20病毒略有过度反应性，而PDK26病毒则过度减弱。我们计划在PDK20克隆中引入进一步的减毒突变，以努力恢复正确的减毒疫苗毒株，首先我们将使用在PDK26中发现的突变的子集，这些突变不在PDK20中。

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.