US3/US7/US8/UL23/UL50基因缺失重组伪狂犬病病毒的构建及对犬致病性分析

Construction of recombinant PRV with US3/US7/US8/UL23/UL50 gene deletion and analysis of its pathogenicity in dogs

自2011年以来,伪狂犬病(PR)在我国猪群中暴发,为控制PR的传播,基于流行的变异株研制了US7/US8/UL23缺失重组伪狂犬病病毒(rPRV)疫苗。疫苗可以对猪提供有效的免疫保护,但由于食用被污染的生肉或免疫猪的内脏,毛皮动物如犬、狐狸、水貂等越来越多地受到PRV弱毒疫苗株的感染,该感染对于毛皮动物是致死性的。本研究以rPRV^(delUS7/US8/UL23)为基础,利用CRISPR/Cas9介导的同源重组技术构建rPRV^(delUS7/US8/UL23/US3/UL50);比较了rPRV^(delUS7/US8/UL23)(三缺失)和rPRV^(delUS7/US8/UL23/US3/UL50)(五缺失)的体外生长动力学和对犬致病性。结果显示,重组病毒rPRV^(delUS7/US8/UL23/US3/UL50)在感染后24~48 h的生长动力学低于rPRV^(delUS7/US8/UL23)。此外,rPRV^(delUS7/US8/UL23)感染细胞可产生细胞融合现象,而^(rPRVdel US7/US8/UL23/US3/UL50)感染细胞未形成细胞融合。US7/US8/UL23缺失rPRV攻毒的犬表现出明显的神经症状,所有犬均死亡,但用US7/US8/UL23/US3/UL50缺失rPRV攻毒的犬未表现出任何神经症状,所有犬在试验期间均存活。组织病毒载量分析并未显示出明显差异。本研究结果表明,US7/US8/UL23/US3/UL50缺失的rPRV对犬无致病力,同时也凸显了US3、UL50基因在PRV感染犬等毛皮动物呈现高毒力中的作用,为研发更安全的疫苗提供了策略。

Since 2011,pseudorabies(PR)outbreak in pigs.To control the spread of pseudorabies,US7/US8/UL23-deleted recombinant pseudorabies virus(rPRV)vaccines based on current variants have been developed.The vaccines can provide effective immune protection to pigs,but fur-bearing animals,such as dogs,foxes,and minks,are increasingly infected by PRV due to consuming contaminated raw meat or offal from immunized pigs.This infection is lethal for these fur animals.The rPRV^(delUS7/US8/UL23/US3/UL50) based on rPRV^(delUS7/US8/UL23) using the CRISPR/Cas9-mediated homologous recombination was constructed.Growth kinetics in vitro and pathogenicity in dogs were compared between rPRV^(delUS7/US8/UL23)(three deletions)and rPRV^(delUS7/US8/UL23/US3/UL50)(five deletions).The results showed that the growth kinetics of rPRV^(delUS7/US8/UL23/US3/UL50) was lower than that of rPRV^(delUS7/US8/UL23) from 24 to 48 h post infection.Moreover,rPRV^(delUS7/US8/UL23)-infected cells formed cell-cell fusion,but the rPRV^(delUS7/US8/UL23/US3/UL50)-infected cells did not.Dogs challenged with US7/US8/UL23-deleted rPRV showed obvious nervous symptoms,and all the dogs died.But the group challenged with the US7/US8/UL23/US3/UL50-deleted rPRV did not show any nervous symptoms,and all the dogs survived for the duration of the experiment.Tissue viral load analyses showed no significant difference in dogs.This study provided evidence that the US7/US8/UL23/US3/UL50-deleted rPRV variant has no pathogenicity to dogs and also highlights the role of the US3and UL50genes in the pathogenicity of PRV in dogs and provides a strategy for developing a safer vaccine.