Heterogeneity and Secondary Structure Analysis of 3' Untranslated Region in Classical Swine Fever Viruses

The attenuated vaccine strains of CSFV have a 12-nucleotides （nt） insertion in the 3＇-UTR of genome as compared to that of CSFV virulent strains. In this study, we found a distinct heterogeneity in the 3＇-UTR of attenuated Thiverval and HCLV strains. The longest 3＇-UTR of Thiverval strain was 259 base pairs （bp） with a 32-nt insertion, the shortest 3＇-UTR had only 233 bp with a 6-nt insertion. The longest 3＇-UTR of HCLV strain was 244 bp with a 17-nt insertion and the shortest 3＇ UTR was 235 bp with a 8-nt insertion. Compared with the published sequences of 3＇-UTR of vaccine and virulent strains, the 3＇-UTR of CSFV vaccine strains have two variable regions where insertion among the different vaccine strains were frequently found. The first is located between the second conservative TALk codon and the start of T-rich region where we found the variable length insertion in the same vaccine strain Thiveral or HCLV and the second is located between the end of T-rich region and the front of GAA eodon, however, a 4-nt deletion was found in this region in the virulent Shimen strain. These two regions may represent the ＂hot spot＂ for mutation. Modeling the secondary structures of the 3＇-UTR suggests that the T-rich insertion could result in the change of structure and free energy, thus affecting the stability of the 3＇-UTR structure. These findings will help to understand the mechanism of attenuated vaccines and improve vaccine safety, stability, and efficacy.

猪瘟病毒3′非编码区的多态性及其二级结构分析

【目的】分析CSFV3′-UTR一级序列和二级结构,为研究其对病毒复制、增殖和毒力的关系奠定良好的基础。【方法】利用RT-PCR技术扩增CSFV Thiverval株、HCLV株和Shimen株的3′-UTR并测序,使用DNAstar、Sequencher和RNAstructure软件进行CSFV3′-UTR基因组序列的比对分析和二级结构模拟。【结果】Thiverval株3′-UTR最长可达259个碱基,与母源株Alfort相比,含有32nt的插入,最短只有233个碱基,含有6nt的插入。HCLV株最长有244个碱基,含有17nt的插入;最短有233个碱基,含有8nt的插入。同时,疫苗株的插入片段导致3′-UTR空间构型和自由能发生改变,自由能随着插入片段的增加而升高。【结论】(1)CSFV3′-UTR存在两个高度变异的区域。(2)CSFV疫苗株Thiverval株和HCLV株3′-UTR同一样品不同克隆株中插入片段呈现明显的不均一。(3)疫苗株的插入片段影响3′-UTR二级结构的稳定性,可能是导致疫苗株毒力减弱的原因之一。