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，也是牛呼吸道综合症的一个组成部分，通常被称为小牛运输热。BoHV-1 还导致小牛流产和奶牛生产力下降。BoHV-1 属于嗜神经性 alphaherpesviruses，它具有感染和建立感觉神经元潜伏期的偏好。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一个有用的平台。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 很容易感染牛源性永生化神经元祖细胞 (FBBC-1)，该细胞在细胞培养物中分化，产生神经突样突起并显示神经元细胞标记 NeuN 和 L1CAM。FBBC-1 细胞在其细胞表面同时表达 nectin-1 和 nectin-2 α疱疹病毒受体，但是，检测到的 nectin-2 的丰度远高于 nectin-1。为了促进 BoHV-1 感染的调查，使用表达绿色荧光蛋白（GFP）的重组 BoHV-1 病毒克隆到细菌人工染色体（BAC）中来生成 mCherry-VP26 融合蛋白。表达 VP26mCherry 的 BoHV-1 GFP 标记病毒感染了分化的 FBBC-1 细胞，感染性病毒体的产生以及 GFP 和 mCherry 荧光团的表达证明了这一点。延时活细胞显微镜显示，在病毒进入以突变方式逆行后，神经元投射中立即存在 mCherry 荧光衣壳。使用 MDBK 细胞的邻近连接测定 (PLA) 证明 BoHV-1 糖蛋白 D (gD) 与 nectin-1 的相互作用比 nectin-2 更有效。然而，与 gD nectin-1 相互作用相比，gD 与 nectin-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 病毒体将有助于实验，旨在阐明病毒在同源牛神经元细胞培养系统中进入和运输的具体机制。使用 MDBK 细胞的邻近连接测定 (PLA) 证明 BoHV-1 糖蛋白 D (gD) 与 nectin-1 的相互作用比 nectin-2 更有效。然而，与 gD nectin-1 相互作用相比，gD 与 nectin-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 病毒体将有助于实验，旨在阐明病毒在同源牛神经元细胞培养系统中进入和运输的具体机制。使用 MDBK 细胞的邻近连接测定 (PLA) 证明 BoHV-1 糖蛋白 D (gD) 与 nectin-1 的相互作用比 nectin-2 更有效。然而，与 gD nectin-1 相互作用相比，gD 与 nectin-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 病毒体将有助于实验，旨在阐明病毒在同源牛神经元细胞培养系统中进入和运输的具体机制。