Innovation Team of Small animal Infectious Disease, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai 200241, China.
Laboratory of Virology, Wageningen University & Research, 6708 PB Wageningen, The Netherlands.
Viruses. 2019 Jun 14;11(6):553. doi: 10.3390/v11060553.
Rabbit haemorrhagic disease virus (RHDV) type 2 (GI.2/RHDV2/b) is an emerging pathogen in wild rabbits and in domestic rabbits vaccinated against RHDV (GI.1). Here we report the genome sequence of a contemporary RHDV2 isolate from the Netherlands and investigate the immunogenicity of virus-like particles (VLPs) produced in insect cells. RHDV2 RNA was isolated from the liver of a naturally infected wild rabbit and the complete viral genome sequence was assembled from sequenced RT-PCR products. Phylogenetic analysis based on the VP60 capsid gene demonstrated that the RHDV2 NL2016 isolate clustered with other contemporary RHDV2 strains. The VP60 gene was cloned in a baculovirus expression vector to produce VLPs in Sf9 insect cells. Density-gradient purified RHDV2 VLPs were visualized by transmission electron microscopy as spherical particles of around 30 nm in diameter with a morphology resembling authentic RHDV. Immunization of rabbits with RHDV2 VLPs resulted in high production of serum antibodies against VP60, and the production of cytokines (IFN-γ and IL-4) was significantly elevated in the immunized rabbits compared to the control group. The results demonstrate that the recombinant RHDV2 VLPs are highly immunogenic and may find applications in serological detection assays and might be further developed as a vaccine candidate to protect domestic rabbits against RHDV2 infection.
兔出血症病毒 2 型（GI.2/RHDV2/b）是一种在野生兔和接种过兔出血症病毒（GI.1）疫苗的家兔中新兴的病原体。本研究报告了来自荷兰的当代 RHDV2 分离株的基因组序列，并研究了在昆虫细胞中产生的病毒样颗粒（VLPs）的免疫原性。从自然感染的野生兔的肝脏中分离出 RHDV2 RNA，并从测序的 RT-PCR 产物中组装了完整的病毒基因组序列。基于 VP60 衣壳基因的系统进化分析表明，RHDV2 NL2016 分离株与其他当代 RHDV2 株聚类在一起。将 VP60 基因克隆到杆状病毒表达载体中，在 Sf9 昆虫细胞中产生 VLPs。通过透射电子显微镜观察，密度梯度纯化的 RHDV2 VLPs 呈现出约 30nm 直径的球形颗粒，形态与真实的 RHDV 相似。用 RHDV2 VLPs 免疫家兔可诱导产生针对 VP60 的高滴度血清抗体，并且与对照组相比，免疫组家兔的细胞因子（IFN-γ 和 IL-4）的产生显著升高。结果表明，重组 RHDV2 VLPs 具有高度的免疫原性，可用于血清学检测，并且可能进一步开发为保护家兔免受 RHDV2 感染的候选疫苗。
