Feline herpesvirus-1 (FHV-1) is the primary cause of viral respiratory and ocular disease in cats. While commercial vaccines can provide clinical protection, they do not protect from infection or prevent latency. Moreover, they are not safe for intranasal administration. Our overall objective is to develop a new mucosal vaccine against FHV-1 disease to address these shortcomings. Feline herpesvirus-1 deletion mutants of glycoprotein C (gC-), gE (gE-), US3-encoded serine/threonine protein kinase (PK-), and both gE and thymidine kinase (gE-TK-) were generated by bacterial artificial chromosome (BAC) mutagenesis. Tracheal tissue explants from eight cats were used to compare the pattern of viral infection and associated tissue damage, as well as virus spread through the basement membrane following inoculation with wild-type virus (WT), and gE-, gE-TK-, PK-, and gC- mutants. Tissues were collected at 24, 48, or 72  hours post-inoculation (hpi) followed by immunohistochemistry (IHC) for FHV-1. Histological changes were graded based on the distribution of virus infected cells and the severity of tissue damage. Inoculations with the WT virus resulted in maximal scores at 72 hpi both at a multiplicity of infection (MOI) of 1 and 0.1. Inoculation with the gE- mutant produced scores similar to scores of explants inoculated with the WT virus at 24 and 48 hpi, but scores were significantly decreased at 72 hpi. Explants inoculated with the gE-TK- mutant showed significantly decreased scores at all time points. Further, the majority of explants inoculated with the PK- mutant resulted in scores of zero at all time points, regardless of MOI. Finally, inoculation with WT resulted in significant stromal invasion below the infected epithelium, while stromal invasion was observed in less than 50 % of the samples following inoculation with gE-, gE-TK-, PK-, or gC- mutants and confined closely to the area surrounding the infected epithelium. In conclusion, the gE-TK- and PK- mutants exhibited significantly reduced virulence, tissue damage and spread to the underlying stroma, suggesting that they may be good vaccine candidates for in vivo testing.

中文翻译：

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组织外植体培养中猫疱疹病毒 1 缺失突变体的表征。

猫疱疹病毒 1 (FHV-1) 是猫病毒性呼吸道和眼部疾病的主要原因。虽然商业疫苗可以提供临床保护，但它们不能防止感染或防止潜伏期。此外，它们对于鼻内给药是不安全的。我们的总体目标是开发一种新的针对 FHV-1 疾病的粘膜疫苗来解决这些缺点。糖蛋白 C (gC-)、gE (gE-)、US3 编码的丝氨酸/苏氨酸蛋白激酶 (PK-) 以及 gE 和胸苷激酶 (gE-TK-) 的猫疱疹病毒 1 缺失突变体由细菌人工产生染色体（BAC）诱变。来自八只猫的气管组织外植体用于比较病毒感染和相关组织损伤的模式，以及在接种野生型病毒 (WT) 和 gE-、gE-TK-、PK- 和 gC- 突变体。在接种后 24、48 或 72 小时 (hpi) 收集组织，然后对 FHV-1 进行免疫组织化学 (IHC)。根据病毒感染细胞的分布和组织损伤的严重程度对组织学变化进行分级。在感染复数 (MOI) 为 1 和 0.1 时，接种 WT 病毒在 72 hpi 时产生最大分数。接种gE-突变体产生的评分与接种WT病毒的外植体在24和48hpi时的评分相似，但评分在72hpi时显着降低。用 gE-TK- 突变体接种的外植体在所有时间点都显示出显着降低的分数。此外，无论MOI如何，接种PK-突变体的大多数外植体在所有时间点都得到零分。最后，用 WT 接种导致受感染上皮下方显着的基质侵袭，而在接种 gE-、gE-TK-、PK- 或 gC- 突变体后，在不到 50% 的样品中观察到基质侵袭，并且紧密局限于该区域围绕受感染的上皮。总之，gE-TK- 和 PK- 突变体表现出显着降低的毒力、组织损伤和向基础基质的扩散，表明它们可能是体内测试的良好候选疫苗。