Department of Pathobiological Sciences and Division of Biotechnology & Molecular Medicine School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, 70803, United States; Division of Biotechnology & Molecular Medicine School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, 70803, United States.
Department of Pathobiological Sciences and Division of Biotechnology & Molecular Medicine School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, 70803, United States; Division of Biotechnology & Molecular Medicine School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, 70803, United States.
Virus Res. 2021 Feb;293:198255. doi: 10.1016/j.virusres.2020.198255. Epub 2020 Dec 15.
Bovine herpesvirus type 1 (BoHV-1) is the viral causative agent of infectious bovine rhinotracheitis and a component of the bovine respiratory complex commonly referred to as shipping fever in calves. BoHV-1 is also responsible for losses of aborted calves and reductions in dairy productivity. BoHV-1 belongs to the neurotropic alphaherpesviruses which have a predilection to infect and establish latency in sensory neurons. Neuronal cell cultures provide a useful platform for experiments investigating neuronal entry, retrograde and anterograde transport, and the establishment of latency. Rodent neuronal cell lines and primary rabbit neuronal cells have been utilized for BoHV-1, though a reliable host-specific neuronal cell culture system has not been developed. In this study, BoHV-1 readily infected bovine-derived immortalized neuronal progenitor cells (FBBC-1) differentiated in cell culture producing neurite-like projections and exhibiting neuronal cell markers NeuN and L1CAM. FBBC-1 cells expressed both nectin-1 and nectin-2 alphaherpesvirus receptors on their cell surfaces, however, nectin-2 was detected in much greater abundance than nectin-1. To facilitate investigations of BoHV-1 infection, a recombinant BoHV-1 virus expressing the green fluorescent protein (GFP) cloned into a bacterial artificial chromosome (BAC) was used to generate an mCherry-VP26 fusion protein. The BoHV-1 GFP expressing VP26mCherry labeled virus infected differentiated FBBC-1 cells as evidenced by the production of infectious virions and the expression of both GFP and mCherry fluorophores. Time-lapse live cell microscopy revealed the presence of mCherry fluorescent capsids in neuronal projections immediately after virus entry moving retrograde in a saltatory manner. Proximity ligation assays (PLA) using MDBK cells demonstrated that BoHV-1 glycoprotein D (gD) interacted more efficiently with nectin-1 than nectin-2. However, the gD interaction with nectin-2 predominated in differentiated FBBC-1 cells in comparison to the gD nectin-1 interaction. The efficiently differentiated FBBC-1 neuronal cell line and fluorescently labeled BoHV-1 virions will assist experimentation aiming to elucidate specific mechanisms of virus entry and transport in a homologous bovine neuronal cell culture system.
牛疱疹病毒 1 型（BoHV-1）是传染性牛鼻气管炎的病毒病原体，也是牛呼吸道疾病复合物的组成部分，在犊牛中通常被称为运输热。BoHV-1 还导致流产牛犊的损失和奶牛生产力的降低。BoHV-1 属于嗜神经α疱疹病毒，其偏好感染和在感觉神经元中建立潜伏。神经元细胞培养物为研究神经元进入、逆行和顺行运输以及潜伏期建立的实验提供了有用的平台。啮齿动物神经元细胞系和原代兔神经元细胞已被用于 BoHV-1，但尚未开发出可靠的宿主特异性神经元细胞培养系统。在这项研究中，BoHV-1 容易感染在细胞培养中分化的牛源永生化神经元祖细胞（FBBC-1），产生类神经突突起，并表现出神经元细胞标志物 NeuN 和 L1CAM。FBBC-1 细胞的细胞表面表达了神经节苷脂 1 和神经节苷脂 2 α疱疹病毒受体，但神经节苷脂 2 的表达量远高于神经节苷脂 1。为了便于研究 BoHV-1 感染，使用克隆到细菌人工染色体 (BAC) 中的绿色荧光蛋白 (GFP) 表达的重组 BoHV-1 病毒产生了 mCherry-VP26 融合蛋白。表达 GFP 的 BoHV-1 VP26mCherry 标记病毒感染分化的 FBBC-1 细胞，证据是产生了感染性病毒粒子，以及 GFP 和 mCherry 荧光团的表达。延时活细胞显微镜观察显示，mCherry 荧光衣壳在病毒进入后立即存在于神经元突起中，以跳跃方式逆行移动。使用 MDBK 细胞的邻近连接分析 (PLA) 表明，BoHV-1 糖蛋白 D (gD) 与神经节苷脂 1 的相互作用效率高于神经节苷脂 2。然而，与 gD 与神经节苷脂 1 的相互作用相比，gD 与神经节苷脂 2 的相互作用在分化的 FBBC-1 细胞中占主导地位。高效分化的 FBBC-1 神经元细胞系和荧光标记的 BoHV-1 病毒粒子将有助于实验，旨在阐明同源牛神经元细胞培养系统中病毒进入和运输的特定机制。
