中文摘要：

通过分析乙型脑炎病毒(Japanese encephalitis virus,JEV)基因组中核糖体移码序列,在对NS2A基因实施同义突变基础上,设计2组引物,通过PCR突变方法,依次删除移码后NS1’基因开放阅读框的终止密码子,并将突变片段替换JEV感染性克隆pJEHEN中相应片段,获得了2株突变全长克隆。经体外转录合成RNA,再以RNA转染细胞,拯救出两株突变病毒vJET1和vJET2。通过RT-PCR扩增突变病毒中含突变位点的基因组片段,测序证实了vJET1基因组3690位核苷酸存在由T向A的突变,vJET2基因组3690位和3819位核苷酸均存在由T向A的突变。将vJET1和vJET2感染Vero细胞后,Western blot检测显示,两株突变病毒表达的NS1’蛋白均出现延长现象。生长曲线显示,与亲本病毒vTHen相比,突变病毒增殖速度均出现下降。动物实验显示,与vTHen相比,vJET1和vJET2对小鼠的神经毒力与神经侵袭力也出现下降。上述结果表明,NS1’编码框的延伸降低病毒在体外培养细胞上的增值速度,也减弱了病毒对小鼠的毒力。

英文摘要：

To study effect of elongated NS1' protein on virulence of Japanese encephalitis virus, two pairs of primers were designed to eliminate stop codons in NS1' gene without amino acid substitutions in NS2A protein. Two full-length mutant clones pJEHENT1 and pJEHENT2 were constructed from the infectious clone of JEV pJEHEN using site-directed mutation of PCR. Sequencing of the rescued viruses vJET1 and vJET2 revealed a T-A mutation at nt 3690 in vJET1 and the same T-A mutations at nt 3690 and nt 3819 in vJET2. Both vJET1 and vJET2 expressed extended NS1' proteins in Vero cells. The growth curves showed that the mutant viruses vJET1 and vJET2 propagated slower than their parent virus vTHEN. Additionally, reduction of neurovirulence and neuroinvasiveness of vJET1 and vJET2 was noted in mouse pathogenecity test. These results indicated that the elongated NS1' protein slowed down growth of the mutant viruses in Vero cells and decreased their virulence to mice.