Nayel Mohamed, Kasem Samy, Fukushi Noriko, El-Habashi Nagwan, Elsify Ahmed, Salama Akram, Hassan Hany, Yanai Tokuma, Ohya Kenji, Fukushi Hideto
Department of Applied Veterinary Sciences, United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
Department of Animal Medicine and Infectious Diseases, Faculty of Veterinary Medicine, University of Sadat City, Sadat City 32897, Egypt.
Pathogens. 2024 Oct 2;13(10):865. doi: 10.3390/pathogens13100865.
Equine herpesvirus type 1 (EHV-1) causes rhinopneumonitis, abortion, and neurological outbreaks (equine herpesvirus myeloencephalopathy, EHM) in horses. EHV-1 also causes lethal encephalitis in small laboratory animals such as mice and hamsters experimentally. EHV-1 ORF76 is a homolog of HSV-1 US9, which is a herpesvirus kinase. Starting with an EHV-1 bacterial artificial chromosome clone of neuropathogenic strain Ab4p (pAb4p BAC), we constructed an ORF76 deletion mutant (Ab4p∆ORF76) by replacing ORF76 with the rpsLneo gene. Deletion of ORF76 had no influence on replication, cell-to-cell spread in cultured cells, or replication in primary neuronal cells. In Western blots of EHV-1-infected cell lysates, an EHV-1 US9-specific polyclonal antibody detected multiple bands ranging from 35 to 42 kDa. In a CBA/N1 mouse infection model following intranasal inoculation, the parent and Ab4p∆ORF76 revertant caused the same histopathology in the brain and olfactory bulbs. The parent, Ab4p∆ORF76, and revertant mutant replicated similarly in the olfactory mucosa, although Ab4p∆ORF76 was not transported to the olfactory bulbs and was unable to infect the CNS. These results indicated that ORF76 (US9) plays an essential role in the anterograde spread of EHV-1.
1型马疱疹病毒（EHV-1）可引发马的鼻肺炎、流产以及神经疾病暴发（马疱疹病毒性脑脊髓炎，EHM）。在实验中，EHV-1还会在小鼠和仓鼠等小型实验动物中引发致命性脑炎。EHV-1的开放阅读框76（ORF76）是单纯疱疹病毒1型（HSV-1）US9的同源物，而HSV-1 US9是一种疱疹病毒激酶。我们以神经致病性毒株Ab4p的EHV-1细菌人工染色体克隆（pAb4p BAC）为起始材料，通过用rpsLneo基因替换ORF76构建了一个ORF76缺失突变体（Ab4p∆ORF76）。ORF76的缺失对病毒复制、在培养细胞中的细胞间传播或在原代神经元细胞中的复制均无影响。在EHV-1感染细胞裂解物的蛋白质免疫印迹分析中，一种EHV-1 US9特异性多克隆抗体检测到了多条分子量在35至42 kDa之间的条带。在鼻内接种后的CBA/N1小鼠感染模型中，亲本毒株和Ab4p∆ORF76回复突变体在大脑和嗅球中引起了相同的组织病理学变化。亲本毒株、Ab4p∆ORF76和回复突变体在嗅黏膜中的复制情况相似，尽管Ab4p∆ORF76未转运至嗅球且无法感染中枢神经系统。这些结果表明，ORF76（US9）在EHV-1的顺行传播中起着至关重要的作用。
