伪狂犬病毒UL14基因缺失突变株的构建及其生物学特性研究

UL14在α-疱疹病毒中相当保守,但其在伪狂犬病毒中的功能尚不清楚。本研究以伪狂犬病毒鄂A株（PRV-Ea）为亲本株,通过同源重组,将编码EGFP的真核表达盒插入到伪狂犬病毒基因组的UL14基因中,PCR检测和荧光观察证实获得了能稳定表达EGFP的UL14插入失活突变株PRV-UL14D。将PRV-UL14D感染IBRS-2细胞,发现该突变株较PRV-Ea野毒株增殖缓慢,而且PRV-UL14D突变株感染细胞后形成的蚀斑也明显小于PRV-Ea野毒株,但在稳定表达UL14的IBRS-2细胞系中,PRV-UL14D与PRV-Ea野毒株的增殖速度、形成的蚀斑大小均无明显差异。进一步将PRV-UL14D感染BALB/c小鼠,发现与相同剂量的PRV-Ea野毒株感染相比,小鼠平均死亡时间明显延缓。上述研究结果表明,UL14并非伪狂犬病毒增殖必需的,但其缺失可延缓病毒增殖,推测UL14可能与病毒粒子在细胞间的扩散有关。

Construction and Characterization of UL14-null Recombinant Pseudorabies Virus

UL14 is a highly conserved protein among alpha-herpesvirus.However,until now,little is known about the function of pseudorabies virus(PRV) UL14.In this study,an UL14-negative PRV mutant(PRV-UL14D) was constructed by inserting an EGFP expression cassette into the UL14 coding sequence.In noncomplementing cells,the mutant was able to replicate,but exhibited an extended growth cycle and significantly reduced plaque sizes,when compared to the field PRV-Ea.These deficiencies were corrected in UL14-expressing cells.In vivo,the average death time of BALB/c mice inoculated with PRV-UL14D was obviously delayed,compared with the same dose of PRV-Ea.From the above results it was therefore concluded that the PRV UL14 product is not essential for virus replication in vitro but delay virus replication and is involved in cell-to-cell spread.