一株A型口蹄疫流行毒株全序列的测定及其全长感染性克隆的构建

Complete genome sequence analysis and construction full-length infectious cDNA clone of an epidemic Foot-and-mouth disease virus of serotype A

摘要:

【目的】测定一株A型口蹄疫流行毒株的全基因组序列，并构建其全长感染性克隆。【方法】参照已公布的A型口蹄疫病毒序列设计引物，将分离的口蹄疫病毒株A/Sea-97/CHA/2014全基因组分为4个重叠的片段进行RT-PCR扩增，并对其进行序列测定与分析。利用酶切连接法将4个基因片段依次克隆至pBluescript SKhdv载体中，构建该流行毒株的全长cDNA克隆pQAHN。pQAHN经Not Ⅰ线性化后转染表达T7 RNA聚合酶的BSR/T7细胞，拯救病毒。【结果】口蹄疫病毒全基因组序列测定结果表明该毒株基因组全长8 171 bp [不包括 poly(C)区段和poly(A)尾巴]，开放阅读框为6 996 bp，编码2 332个氨基酸，5′和3′非编码区分别为1 091 bp和95 bp。VP1系统发生树分析表明该毒株与A/GDMM/CHA/2013毒株亲缘关系最近，相似性为99.1%。线化全长质粒转染BSR/T7细胞68 h后可观察到典型的细胞病变。拯救病毒的间接免疫荧光、RT-PCR和序列测定结果表明成功拯救出了具有感染性的FMDV。拯救病毒与亲本病毒的噬斑表型及生长曲线试验表明二者具有相似的生长表型和增殖能力。【结论】该研究为我国口蹄疫病原生态分布、分子流行病学调查以及A型FMD新型疫苗的研究提供了有益的材料。

Abstract:

[Objective] Sequencing and analysis complete genome of an epidemic foot-and-mouth disease virus of serotype A and construction of its full-length infectious cDNA clone. [Methods] The primers were designed according to the published genome sequence of type A FMDV and a total of four fragments covering the complete genome of A/Sea-97/CHA/2014 were subsequently PCR amplified and sequenced. The four fragments were cloned into pBluescript SKhdv vector in turn with a single restriction sites to construct a full-length cDNA clone of A/Sea-97/CHA/2014 strain. The full-length plasmid linearized with Not Ⅰ and transfected to BSR/T7 cells expressing T7 RNA polymerase to rescue the recombinant virus. [Results] The result of sequence analysis showed that FMDV A/Sea-97/CHA/2014 strain was 8 171 bp in length [except poly(C) and poly(A)], which contains a 5′-UTR with 1 091 bp, a 3′-UTR with 95 bp and a ORF encoding 2 332 amino acids with 6 996 bp. The phylogenetic tree based on the nucleotide sequences of VP1 gene revealed that the A/Sea-97/CHA/2014 strain had a high sequence identity with A/GDMM/CHA/2013 strain (99.1%). The full-length plasmid transfected to BSR/T7 cells, apparent CPE were observed after 68 h incubation. The harvested virus was verified by IFA, RT-PCR and sequencing. The results indicated that infectious FMDV was successfully rescued in vivo. Plaque phenotype and growth curves tests of rescue virus and parental virus showed they have a similar growth phenotype and capacity of proliferation. [Conclusion] This study provided a useful material for studies of the FMD pathogen ecological distribution, molecular epidemiological as well as novel marker vaccines.