Chen W, Kawano H, Men R, Clark D, Lai C J
Molecular Viral Biology Section, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892, USA.
J Virol. 1995 Aug;69(8):5186-90. doi: 10.1128/JVI.69.8.5186-5190.1995.
There are four dengue virus serotypes (DEN1 to -4), each of which causes major epidemics in tropical or subtropical areas. The current strategy for dengue virus immunization favors the use of a tetravalent vaccine preparation. We have previously employed full-length DEN4 cDNA to construct a viable intertypic dengue virus type 1 or type 2 chimera that contained the C-PreM-E or only the PreM-E genes of DEN1 or DEN2 substituting for the corresponding genes of DEN4. This success implied that it might be possible to create mutants of all four dengue virus serotypes for evaluation as candidate vaccines. In this study, we constructed DEN3-DEN4 chimeras that contained DEN3 C-PreM-E genes and expressed DEN3 antigenic specificity. Unlike our previous successes in cloning DEN1 or DEN2 chimeric cDNA, we were not able to clone the DEN3 C-PreM-E genes directly in the 5' intermediate vector or in the full-length chimeric DEN3-DEN4 plasmid in Escherichia coli. Nevertheless, a full-length DNA template of DEN3-DEN4 that could be used for transcription of infections RNAs was prepared by in vitro ligation. Progeny virus recovered from RNA-transfected C6/36 mosquito cells exhibited DEN3 antigenic specificity as determined by a reaction with monoclonal antibodies. Gel electrophoresis of virus-infected cell lysates yielded the predicted viral protein pattern, i.e., DEN3 C, PreM, and E and DEN4 nonstructural proteins. Two amino acid substitutions, Thr-435-->Leu and Glu-406-->Lys, which are analogous to mutations that, respectively, confer mouse neurovirulence on DEN4 and DEN2, were introduced into DEN3 E. A mutant chimera containing the Thr-435-->Leu substitution, which ablates the potential glycosylation site sequence, produced an E protein identical in size to that of wild-type DEN3 E, indicating that the glycosylation site is normally not used. Intracerebral inoculation of suckling mice revealed that the mutant chimera containing the Glu-406-->Lys substitution was neurovirulent, whereas its wild-type counterpart or parent DEN3 was not.
登革病毒有四种血清型（DEN1至DEN4），每种血清型都会在热带或亚热带地区引发大规模疫情。目前登革病毒免疫的策略倾向于使用四价疫苗制剂。我们之前利用全长DEN4 cDNA构建了一种可行的1型或2型跨血清型登革病毒嵌合体，该嵌合体包含DEN1或DEN2的C-PreM-E基因或仅PreM-E基因，取代了DEN4的相应基因。这一成功意味着有可能构建出所有四种登革病毒血清型的突变体，作为候选疫苗进行评估。在本研究中，我们构建了包含DEN3 C-PreM-E基因并表达DEN3抗原特异性的DEN3-DEN4嵌合体。与我们之前成功克隆DEN1或DEN2嵌合cDNA不同，我们无法在5'中间载体或大肠杆菌中的全长嵌合DEN3-DEN4质粒中直接克隆DEN3 C-PreM-E基因。然而，通过体外连接制备了可用于转录感染性RNA的DEN3-DEN4全长DNA模板。从RNA转染的C6/36蚊细胞中回收的子代病毒通过与单克隆抗体反应显示出DEN3抗原特异性。病毒感染细胞裂解物的凝胶电泳产生了预测的病毒蛋白模式，即DEN3的C、PreM和E以及DEN4的非结构蛋白。将两个氨基酸替换，Thr-435→Leu和Glu-406→Lys，引入DEN3 E，这两个替换分别类似于使DEN4和DEN2具有小鼠神经毒力的突变。一个包含Thr-435→Leu替换的突变嵌合体消除了潜在的糖基化位点序列，产生了与野生型DEN3 E大小相同的E蛋白，表明该糖基化位点通常未被使用。对乳鼠进行脑内接种显示，包含Glu-406→Lys替换的突变嵌合体具有神经毒力，而其野生型对应物或亲本DEN3则没有。
