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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 by with a 6-nt insertion.The longest 3'-UTR of HCLV strain was 244 by with a 17-nt insertion and the shortest 3'UTR was 235 by 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 TAA 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 codon,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'-UTRsuggests that the Trich 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.