口蹄疫O型重组病毒的构建及其免疫原性分析

【目的】利用反向遗传操作技术，构建含O型口蹄疫病毒(food-and-mouth disease virus, FMDV) 3个拓扑型免疫优势结构蛋白基因的重组FMDV，评估其作为猪O型口蹄疫(food-and-mouth disease, FMD)疫苗候选株的潜力。【方法】通过基因合成，在FMD疫苗株O/HN/CHA/93 (古典中国拓扑型)的基因中嵌合流行株O/NXYCh/CHA/2018 (东南亚拓扑型) VP1结构蛋白的重组病毒骨架上，用O/TUR/5/2009疫苗株(中东-南亚拓扑型) VP1蛋白的G-H环基因替换其对等基因，构建含O型3个拓扑型FMDV结构蛋白基因的重组全长质粒，Not I线性化后转染表达T7 RNA聚合酶的BSR/T7细胞，拯救重组病毒。通过RT-PCR、序列测定、间接免疫荧光鉴定重组病毒；噬斑试验和一步生长曲线分析重组病毒的生物学特性。重组病毒制备疫苗免疫猪，用病毒中和试验分析其对当前流行的O型3个拓扑型FMDV的交叉反应性。【结果】成功拯救到含O型3个拓扑型FMDV结构蛋白基因的重组病毒，重组病毒与亲本病毒具有相似的生物学特性。亲本病毒和重组病毒制备的疫苗免疫猪，均能够对中东-南亚型(Middle East-South Asia, ME-SA)拓扑型和东南亚型(South-East Asia, SEA)拓扑型病毒株产生保护性平均中和抗体(>1.65log₁₀)；均不能对古典中国型(Cathay)拓扑型流行株产生保护性平均中和抗体(<1.65log₁₀)，但与亲本病毒相比，O/TUR/5/2009疫苗株G-H环基因的替换显著提高了对ME-SA和SEA拓扑型病毒株的交叉反应性(p<0.05)。【结论】本研究对未来FMD疫苗的设计具有重要的指导意义。

Construction and immunogenicity of serotype O recombinant foot-and-mouth disease virus

[Objective] To construct a recombinant food-and-mouth disease virus (FMDV) strain carrying the genes encoding three topotypes of immunodominant structural proteins of serotype O FMDV by reverse genetic manipulation and evaluate the potential of the recombinant strain serving as a vaccine candidate for porcine food-and-mouth disease (FMD) type O. [Methods] Based on the gene of the recombinant FMDV with the replacement of the VP1 structural protein of O/NXYCh/CHA/2018 epidemic strain, the recombinant full-length plasmid featuring substitution of G-H loop genes of the structural protein VP1 of O/TUR/5/2009 vaccine strain was constructed by gene synthesis. The recombinant virus was rescued after transfection of the linearized recombinant plasmid into BSR/T7 cells expressing T7 RNA polymerase, and then identified by RT-PCR, sequencing, and indirect immunofluorescence. The plaque assay and one-step growth curve building were employed to characterize the recombinant virus. Pigs were vaccinated with the vaccines prepared from the recombinant virus and the parental virus, and then virus neutralization tests were carried out to examine the cross-reactive responses against the epidemic serotype O FMDV isolates of three topotypes. [Results] The recombinant FMDV strain carrying the structural protein genes of three topotypes was successful rescued. The recombinant strain showed similar biological properties to the parental virus. Pigs vaccinated with the vaccines prepared from the recombinant virus and the parental virus produced protective neutralizing antibodies with the mean titer of >1.65log₁₀ against the viruses of the Middle East-South Asia (ME-SA) and South-East Asia (SEA) topotypes. The pigs did not produce protective neutralizing antibodies against the Cathay topotype (<1.65log₁₀). The substitution of O/TUR/5/2009 G-H loop gene improved the cross-reactivity against the viruses of ME-SA and SEA topotypes compared with the parental virus (p<0.05). [Conclusion] This study has guiding significance for the design of FMD vaccines in the future.