Wang A P, Liu L, Gu L L, Wu S, Guo C M, Feng Q, Xia W L, Yuan C, Zhu S Y
Acta Virol. 2019;63(1):53-59. doi: 10.4149/av_2019_104.
The avian adeno-associated virus (AAAV) is a replication-defective nonpathogenic virus that has been proved to be useful as a viral vector in gene delivery. In this study, the feasibility of AAAV for transgenic expression of duck hepatitis A virus (DHAV) VP3 structural protein and its ability to induce protective immunity in ducklings was assessed. The recombinant AAAV (rAAAV-VP3) expressing the VP3 protein was prepared by co-infection of Sf9 cells with recombinant baculovirus (rBac-VP3) containing VP3 gene flanked by inverted terminal repeats (ITRs) of AAAV and the other two recombinant baculovirus expressing AAAV functional and structural genes, respectively. The generation of rAAAV-VP3 was demonstrated by electron microscopy, immunofluorescence assay, and western blot analysis. One day old ducklings were inoculated with rAAAV-VP3 or commercial attenuated vaccine and then challenged with DHAV-1 strain SH two weeks post vaccination. Anti-DHAV-1 antibodies were detected in all vaccinated groups by ELISA, and the titers between the rAAAV-VP3 group and the attenuated vaccine group were not statistically significant. Real time RT-PCR analysis showed that the virus copy numbers in the livers of the PBS control group were significantly higher than that of the rAAAV-VP3 and attenuated vaccine groups. In conclusion, we demonstrated that the VP3 expression mediated by rAAAV in ducklings could induce protective immunity against DHAV challenge, and this could be a candidate vaccine for the control of duck viral hepatitis. Keywords: avian adeno-associated virus; duck hepatitis A virus; VP3 gene; immunogenicity.
禽腺相关病毒（AAAV）是一种复制缺陷型非致病性病毒，已被证明可作为基因递送的病毒载体。在本研究中，评估了AAAV用于鸭甲型肝炎病毒（DHAV）VP3结构蛋白转基因表达的可行性及其在雏鸭中诱导保护性免疫的能力。通过将含有AAAV反向末端重复序列（ITR）侧翼的VP3基因的重组杆状病毒（rBac-VP3）与另外两种分别表达AAAV功能和结构基因的重组杆状病毒共同感染Sf9细胞，制备了表达VP3蛋白的重组AAAV（rAAAV-VP3）。通过电子显微镜、免疫荧光测定和蛋白质印迹分析证实了rAAAV-VP3的产生。1日龄雏鸭接种rAAAV-VP3或商业减毒疫苗，然后在接种后两周用DHAV-1株SH进行攻毒。通过ELISA在所有接种组中检测到抗DHAV-1抗体，rAAAV-VP3组和减毒疫苗组之间的抗体滴度无统计学差异。实时RT-PCR分析表明，PBS对照组肝脏中的病毒拷贝数显著高于rAAAV-VP3组和减毒疫苗组。总之，我们证明rAAAV介导的VP3在雏鸭中表达可诱导针对DHAV攻击的保护性免疫，这可能是一种用于控制鸭病毒性肝炎的候选疫苗。关键词：禽腺相关病毒；鸭甲型肝炎病毒；VP3基因；免疫原性。
