State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China.
Key Laboratory of Preventive Veterinary Medicine in Hubei Province, Cooperative Innovation Center for Sustainable Pig Production, Wuhan, 430070, China.
Virol J. 2020 Feb 3;17(1):18. doi: 10.1186/s12985-020-1288-4.
Porcine β-defensin 2 (PBD-2), produced by host cells, is an antimicrobial cysteine-rich cationic peptide with multi-functions. Previous studies have demonstrated that PBD-2 can kill various bacteria, regulate host immune responses and promote growth of piglets. However, the antiviral role of PBD-2 is rarely investigated. This study aimed to reveal the antiviral ability of PBD-2 against pseudorabies virus (PRV), the causative pathogen of Aujeszky's disease, in PK-15 cells and in a PBD-2 expressing transgenic (TG) mouse model.
In this study, the cytotoxicity of PBD-2 on PK-15 cells was measured by CCK-8 assay. PK-15 cells were incubated with PRV pre-treated with different concentrations of PBD-2 and PRV titers in cell culture supernatants were determined by real-time quantitative PCR (RT-qPCR). TG mice and wild-type (WT) mice were intraperitoneally injected with PRV and the survival rate was recorded for 10 days. Meanwhile, tissue lesions in brain, spleen and liver of infected mice were observed and the viral loads of PRV in brain, liver and lung were analyzed by RT-qPCR.
PBD-2 at a maximum concentration of 80 μg/mL displayed no significant cytotoxicity on PK-15 cells. A threshold concentration of PBD-2 at 40 μg/mL was required to inhibit PRV proliferation in PK-15 cells. The survival rate in PBD-2 TG mice was 50% higher than that of WT mice. In addition, TG mice showed alleviated tissue lesions in brain, spleen and liver compared with their WT littermates after PRV challenge, while viral loads of PRV in brain, liver and lung of TG mice were significantly lower than that of WT mice.
PBD-2 could inhibit PRV proliferation in PK-15 cells and protect mice from PRV infection, which confirmed the antiviral ability of PBD-2 both in vitro and in vivo. The application of PBD-2 in developing anti-viral drugs or disease-resistant animals can be further investigated.
宿主细胞产生的猪β防御素 2（PBD-2）是一种具有多种功能的抗菌含半胱氨酸阳离子肽。先前的研究表明，PBD-2 可以杀死各种细菌，调节宿主免疫反应并促进仔猪生长。但是，PBD-2 的抗病毒作用很少被研究。本研究旨在揭示 PBD-2 对伪狂犬病病毒（PRV）的抗病毒能力，PRV 是猪水疱病的病原体，在 PK-15 细胞和 PBD-2 表达转基因（TG）小鼠模型中。
在这项研究中，通过 CCK-8 测定法测量 PBD-2 对 PK-15 细胞的细胞毒性。用不同浓度的 PBD-2 预处理 PRV 后孵育 PK-15 细胞，并通过实时定量 PCR（RT-qPCR）测定细胞培养上清液中的 PRV 滴度。通过腹腔内注射 PRV 对 TG 小鼠和野生型（WT）小鼠进行感染，并记录 10 天的存活率。同时，观察感染小鼠脑，脾和肝的组织病变，并通过 RT-qPCR 分析 PRV 在脑，肝和肺中的病毒载量。
PBD-2 的最大浓度为 80μg/mL 对 PK-15 细胞没有明显的细胞毒性。在 PK-15 细胞中抑制 PRV 增殖需要 40μg/mL 的 PBD-2 阈值浓度。与 WT 小鼠相比，PBD-2 TG 小鼠的存活率提高了 50％。此外，与 WT 同窝仔相比，PRV 攻击后 TG 小鼠的脑，脾和肝的组织病变减轻，而 TG 小鼠的脑，肝和肺中的 PRV 病毒载量明显低于 WT 小鼠。
PBD-2 可以抑制 PK-15 细胞中 PRV 的增殖并保护小鼠免受 PRV 感染，这证实了 PBD-2 在体外和体内的抗病毒能力。可以进一步研究 PBD-2 在开发抗病毒药物或抗病动物中的应用。
