Institute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institut, Südufer 10, 17493 Greifswald, Isle of Riems, Germany.
Department of Experimental Animal Facilities and Biorisk Management, Friedrich-Loeffler-Institut, Südufer 10, 17493 Greifswald, Insel Riems, Germany.
J Immunol Methods. 2018 Sep;460:36-44. doi: 10.1016/j.jim.2018.06.006. Epub 2018 Jun 9.
Rift Valley fever virus is a mosquito-borne virus which is associated with acute hemorrhagic fever leading to large outbreaks among ruminants and humans in Africa and the Arabian Peninsula. RVFV circulates between mosquitoes, ruminants, camels and humans, which requires divergent amplification and maintenance strategies that have not been fully explored on the cellular and molecular level. We therefore assessed monoclonal antibodies for their applicability to monitor the expression pattern and kinetics of viral proteins in different RVFV infected cell species. Sequences of RVFV vaccine strain MP-12 were used in a bacterial expression system to produce recombinant non-structural proteins directed to NSs and NSm. After immunization of balb/c mice a set of monoclonal antibodies were generated and extensively characterized. The kinetics of RVFV proteins in vertebrate (Vero76) and mosquito-derived (C6/36) cells were evaluated with monoclonal antibodies against the nucleocapsid protein (NP) and the glycoproteins (Gn and Gc) as well as with the newly generated NSs and NSm derived monoclonal antibodies. Significant differences of viral protein distribution and accumulation in vertebrate compared to mosquito-derived cells could be demonstrated. Differences were observed for the nonstructural NSm and most intriguingly for the NSs protein indicating significant divergency of replication strategies of RVFV in Vero 76 cells and C6/36 cells. The described monoclonal antibodies are therefore powerful tools to elucidate the discrepancies of virus replication and interaction within the mammalian host compared to the mosquito vector.
裂谷热病毒是一种蚊媒病毒，与急性出血热有关，在非洲和阿拉伯半岛的反刍动物和人类中引发大规模疫情。RVFV 在蚊子、反刍动物、骆驼和人类之间循环，这需要不同的扩增和维持策略，这些策略在细胞和分子水平上尚未得到充分探索。因此，我们评估了单克隆抗体在监测不同 RVFV 感染细胞物种中病毒蛋白表达模式和动力学方面的适用性。使用 RVFV 疫苗株 MP-12 的序列在细菌表达系统中产生针对 NSs 和 NSm 的重组非结构蛋白。用免疫 Balb/c 小鼠产生了一组单克隆抗体，并对其进行了广泛的表征。用针对核衣壳蛋白 (NP) 和糖蛋白 (Gn 和 Gc) 的单克隆抗体以及新产生的 NSs 和 NSm 衍生的单克隆抗体评估了 RVFV 蛋白在脊椎动物 (Vero76) 和蚊子衍生 (C6/36) 细胞中的动力学。与蚊子衍生的细胞相比，在脊椎动物中可以证明病毒蛋白分布和积累的显著差异。在非结构 NSm 中观察到差异，最有趣的是在 NSs 蛋白中观察到差异，表明 RVFV 在 Vero 76 细胞和 C6/36 细胞中的复制策略存在显著差异。因此，所描述的单克隆抗体是阐明病毒在哺乳动物宿主内与蚊子媒介之间的复制差异和相互作用的有力工具。
