Gao Ting, Gao Caixia, Wu Siyu, Wang Yingying, Yin Jiyuan, Li Yingying, Zeng Weiwei, Bergmann Sven M, Wang Qing
Key Lab of Fishery Drug Creation of Ministry of Agriculture and Rural Areas, Key Lab of Aquatic Animal Immune Technology of Guangdong Province, Pearl River Fisheries Research Institute, Chinese Academy of Fishery Science, Guangzhou 510380, China.
Department of Aquatecture, Tianjin Agricultural University, Tianjin 300384, China.
Vaccines (Basel). 2021 Jan 14;9(1):53. doi: 10.3390/vaccines9010053.
Grass carp reovirus (GCRV) leads to severe hemorrhagic disease in grass carp () and causes economic losses in grass carp aquaculture. Recent epidemiological investigations showed that GCRV genotype II is the dominant subtype in China. Therefore, it is very important to develop a novel vaccine for preventing diseases caused by GCRV genotype II. In this study, we employed a bac-to-bac expression system to generate GCRV-II-based virus-like particles (VLPs). Previous studies have shown that the structural proteins VP3, VP4, and VP38 encoded by the segments S3, S6, and S10 of type II GCRV are immunogenic. Hence, the GCRV-VLPs were produced by co-infection of sf9 cells with recombinant baculoviruses PFBH-VP3, PFBH-VP4, and PFBH-VP38. The expressions of VP3, VP4, and VP38 proteins in GCRV-VLPs were tested by IFA and Western blot analysis. By electron microscopic observations of ultrathin sections, purified VLPs showed that the expressed proteins are similar in shape to GCRV genotype II with a size range from 40 nm to 60 nm. The immunogenicity of GCRV-VLPs was evaluated by the injection immunization of grass carp. The analysis of serum-specific IgM antibody showed that grass carp immunized with GCRV-VLPs produced GCRV-specific antibodies. Furthermore, injection with GCRV-VLPs increased the expressions of immune-related genes (IgM, IFN, TLR3, TLR7) in the spleen and kidney. In addition, grass carp immunized with a GCRV-VLPs-based vaccine showed a relative percent survival rate (RPS) of 83.33% after challenge. The data in this study showed that GCRV-VLPs demonstrated an excellent immunogenicity and represent a promising approach for vaccine development against GCRV genotype II infection.
草鱼呼肠孤病毒（GCRV）可导致草鱼严重出血性疾病，给草鱼养殖业造成经济损失。近期的流行病学调查表明，GCRV基因型II是中国的主要亚型。因此，开发一种新型疫苗来预防由GCRV基因型II引起的疾病非常重要。在本研究中，我们采用杆状病毒表达系统来生产基于GCRV-II的病毒样颗粒（VLPs）。先前的研究表明，II型GCRV的S3、S6和S10片段编码的结构蛋白VP3、VP4和VP38具有免疫原性。因此，通过用重组杆状病毒PFBH-VP3、PFBH-VP4和PFBH-VP38共感染sf9细胞来生产GCRV-VLPs。通过免疫荧光分析（IFA）和蛋白质免疫印迹分析（Western blot）检测GCRV-VLPs中VP3、VP4和VP38蛋白的表达。通过对超薄切片的电子显微镜观察，纯化的VLPs显示表达的蛋白在形状上与GCRV基因型II相似，大小范围为40纳米至60纳米。通过对草鱼进行注射免疫来评估GCRV-VLPs的免疫原性。血清特异性IgM抗体分析表明，用GCRV-VLPs免疫的草鱼产生了GCRV特异性抗体。此外，注射GCRV-VLPs增加了脾脏和肾脏中免疫相关基因（IgM、IFN、TLR3、TLR7）的表达。此外,用基于GCRV-VLPs的疫苗免疫的草鱼在攻毒后显示出83.33%的相对存活率（RPS）。本研究的数据表明，GCRV-VLPs具有优异的免疫原性，是开发针对GCRV基因型II感染的疫苗的一种有前景的方法。
