The G-H loop of the foot-and-mouth disease virus（FMDV） virion contains certain dominant immunogenic epitopes, as well as an arginine-glycine-aspartic acid（RGD） motif that is recognized by cell surface integrin rec...The G-H loop of the foot-and-mouth disease virus（FMDV） virion contains certain dominant immunogenic epitopes, as well as an arginine-glycine-aspartic acid（RGD） motif that is recognized by cell surface integrin receptors. Previous experiments indicate that it is critical to maintain virus structural integrity when inserting an exogenous epitope into the surface of an FMDV structural protein. However, it remains to be determined how factors such as different insertion positions affect interactions among the virus, cells and host immune system. In this study, one infectious c DNA clone of the swine FMDV Cathay topotype strain O/CHA/90 was constructed. Then, a FLAG marker（DYKDDDDK） was inserted upstream（–4） or downstream（＋10） of the RGD motif to generate tagged viruses vFLAG-O/CHA/90 or vO/CHA/90-FLAG, investigating the possibility of expressing foreign antigen and effect on its immunogenicity. Compared to the parental virus, both tagged viruses exhibited similar plaque phenotypes, suckling mouse pathogenicity and antigenicity. Additionally, the FLAGtag insertion position did not change the use of integrin-mediated cell entry by the tagged viruses. Interestingly, both tagged vaccines protected pigs against challenge with the parental virus O/CHA/90 and induced immune responses against FMDV in BALB/c mice and pigs, but only vaccination with vFLAG-O/CHA/90 generated anti-FLAG antibodies. Our findings demonstrated that two sites（RGD–4 and RGD＋10） tolerated the insertion of an exogenous gene in the swine FMDV O/CHA/90 strain. However, only RGD–4 was a novel and appropriate inserting site which could tolerate exogenous FLAG. The resultant tagged virus is a promising candidate for FMD vaccine which can be differentiating infected from vaccinated animals（DIVA）.展开更多
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 a...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.展开更多
基金supported by the National Key Research and Development Program of China(2016YFD0501500)the Special Fund for Agro-scientific Research in the Public Interest,China(201303046)
文摘The G-H loop of the foot-and-mouth disease virus（FMDV） virion contains certain dominant immunogenic epitopes, as well as an arginine-glycine-aspartic acid（RGD） motif that is recognized by cell surface integrin receptors. Previous experiments indicate that it is critical to maintain virus structural integrity when inserting an exogenous epitope into the surface of an FMDV structural protein. However, it remains to be determined how factors such as different insertion positions affect interactions among the virus, cells and host immune system. In this study, one infectious c DNA clone of the swine FMDV Cathay topotype strain O/CHA/90 was constructed. Then, a FLAG marker（DYKDDDDK） was inserted upstream（–4） or downstream（＋10） of the RGD motif to generate tagged viruses vFLAG-O/CHA/90 or vO/CHA/90-FLAG, investigating the possibility of expressing foreign antigen and effect on its immunogenicity. Compared to the parental virus, both tagged viruses exhibited similar plaque phenotypes, suckling mouse pathogenicity and antigenicity. Additionally, the FLAGtag insertion position did not change the use of integrin-mediated cell entry by the tagged viruses. Interestingly, both tagged vaccines protected pigs against challenge with the parental virus O/CHA/90 and induced immune responses against FMDV in BALB/c mice and pigs, but only vaccination with vFLAG-O/CHA/90 generated anti-FLAG antibodies. Our findings demonstrated that two sites（RGD–4 and RGD＋10） tolerated the insertion of an exogenous gene in the swine FMDV O/CHA/90 strain. However, only RGD–4 was a novel and appropriate inserting site which could tolerate exogenous FLAG. The resultant tagged virus is a promising candidate for FMD vaccine which can be differentiating infected from vaccinated animals（DIVA）.
基金supported by the National Natural Science Foundation of China (30571377)the National High-Tech R&D Program of China (863 Program,2006AA10A204)
文摘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.
