伪狂犬病病毒变异株神经毒力增强的分子机制

Since late 2011, there have been pseudorabies outbreaks in a large number of Bartha-K61-vaccinated swine herds in many regions of China. Epidemiological studies showed that a pseudorabies virus (PRV) variant is responsible for the outbreaks and the currently used Bartha-K61 vaccine cannot provide complete protection against the emerging PRV variant. Our previous study indicated that the PRV variant TJ strain is more pathogenic than previous PRV isolates (including the highly virulent SC strain) and is able to induce unexpected pruritus in pigs. Considering the correlation between pruritus, virulence, and neuroinvasiveness, the aim of this proposal is to evaluate the directional spread of chimeric PRV mutants derived from the PRV SC strain and the TJ strain in the nervous system. The PRV mutants will be generated by using PRV bacterial artificial chromosome and CRISPR/Cas9 system on the basis of the genomic sequence alignment between the TJ and SC strains. Thereafter, the directional infection and spread of the mutants and parent strains in neurons will be evaluated using a tri-chambered neuron culturing system and fluorescent fusion proteins in conjunction with time-lapse microscopy, and the pathogenicity of the PRV mutants and parent strains will be compared in mice and pigs. This proposal will be expected to illuminate the molecular mechanisms of enhanced neurovirulence of the PRV variant. This study will provide insight into the pathogenesis and vaccine design for the PRV variant, and also provide a model for study of the spread mechanisms of other neuroinvasive viruses.

2011年以来，我国多个地区免疫过伪狂犬病弱毒疫苗Bartha-K61株的猪场爆发了伪狂犬病。研究表明，此轮疫情是由伪狂犬病病毒（PRV）变异株引起。此前我们研究发现，PRV变异株的致病性明显增强，猪感染后出现前所未见的瘙痒症状。鉴于PRV感染所引起的瘙痒及致病性增强与其对神经系统的侵袭有直接的关系，本项目拟以PRV经典强毒株（SC株）和变异株（TJ株）为背景，利用PRV细菌人工染色体和CRISPR/Cas9基因组编辑技术，构建一系列神经毒力相关基因互换的嵌合病毒；利用体外神经元三室培养系统、荧光蛋白融合表达结合时差显微成像技术等，系统评价相应嵌合病毒及其亲本毒株在神经元中传导及其对小鼠和猪只致病性的差异，从而阐明PRV变异株引起感染猪只瘙痒和致病力增强的分子机制。本项目将为PRV变异株致病机制研究和疫苗设计提供科学数据，并为其它神经侵袭性病毒在神经系统中传导机制的研究提供借鉴。

伪狂犬病病毒（PRV）变异株毒力增强，且感染猪只后引起前所未有的瘙痒症状，传统疫苗株Bartha-K61免疫后不能对PRV变异株提供完全保护，PRV变异株毒力增强的机制不明确。本项目从体内外两方面比较PRV变异株（TJ）和经典株（SC）毒力差异，并分析其毒力差异的机制。首先为了便于快速构建PRV突变株，先后建立了PRV TJ和PRV SC的Fosmid系统；利用此操作系统分别构建了rPRVTJ-UL36-EGFP和rPRVSC-UL36-EGFP重组病毒；经鉴定这两株报告病毒遗传稳定，形态和生长特征与亲本毒相似；将rPRVTJ-UL36-EGFP和rPRVSC-UL36-EGFP以107 TCID50分别感染微流体中神经元胞体和轴突，经测定rPRVTJ-UL36-EGFP逆轴突传导效率高于rPRVSC-UL36-EGFP，两毒株在轴突中逆向传导的速率差异不显著。而顺轴突传导效率差异不明显。其次，将PRV TJ和PRV SC以103 TCID50的剂量经肌肉注射途径接种小鼠，之后通过观察小鼠的临床症状、记录小鼠的体质量变化和死亡率，利用荧光定量PCR（qPCR）检测小鼠各组织中病毒基因组的拷贝数，以及分析小鼠各组织脏器的病理学变化来系统评价PRV变异株和经典株的致病力差异。研究结果显示，接种PRV TJ和PRV SC的小鼠在临床表现上无明显差异，但接种PRVTJ的小鼠体质量下降明显，死亡率相对更高；qPCR检测结果显示，PRVTJ对小鼠的组织嗜性增强，尤其是对脑组织的嗜性；病理组织学分析表明，接种PRVTJ的小鼠脑组织的炎性病变更明显。本研究证实，PRVTJ对小鼠的致病力增强，且其毒力增强的主要原因是对小鼠的神经嗜性增强。