Construction And Characteristics Analysis Of Genetically Engineered Type O Foot-and-mouth Disease Virus With Acid Stability

Foot-and-mouth disease(FMD) caused by foot-and-mouth disease virus(FMDV) is an extremely contagious disease of cloven-hoofed animals and economically devastating disease affecting animal agriculture and international trade throughout the world. The whole virus(146S) of FMDV is the most effective viral antigen which is used to produce inactivated vaccine. 146 S is highly acid labile and dissociates into pentameric subunits in environment slightly below neutrality, decreasing the content of effective antigens and reducing the efficacy of vaccines. To develop the vaccines candidate strain with improved acid stability, prove if the substitution VP2D86 H could confer acid stability to FMDV and study the molecular determinants associated with the acid-resistant phenotype of acid stable FMDV, six mutants with single or combined amino acid substitutions in capsid protein(VP1N17D, VP2H145 Y, VP2D86 H, VP3H141 D, VP3H141 G, and VP1N17D+VP2H145Y)were constructed based on a recently developed type O FMDV infectious cDNA clone and recovered in vivo after transfecting the BSR/T7 cells.The observation of CPE and indirect immunofluorescent assay demonstrated that the four FMDVs respectively including the substitution VP1N17 D, VP2D86 H, VP2H145 Y and VP1N17D+VP2H145Y were rescued and the substitution VP3H141G/D was lethal for parent virus. RT-PCR and sequencing proved that the recovered virus rN17 D containing the substitution VP1N17 D and rD86 H including the substitution VP2D86 H were stably inheritable and the rescued virus rH145 Y containing the substitution VP2H145 Y and rN17D2 including the substitution VP1N17D+VP2H145Y both occurred reverse mutation and compensatory mutations from first generation. A number of characteristics of the rescued virus rN17 D, rD86 H and rN17D2 were subsequently analysed. The results indicated that amino acid VP1N17 D and VP2D86 H respectively increased the acid, thermal and alkaline resistance of parental FMDV O/HN/93. However, the mutant rD86 H displayed higher replication ability and stronger suckling mice virulence than the mutant rN17 D. Serum-neutralising titre of the two rescued FMDVs was also extremely discrepant because of the significantly different spatial distance between antigenic site 1 and 5. This is the first time that we demonstrate the substitution VP2 D86 H can confer acid stability and thermal stability, we also prove that the FMDV rD86 H is better than the FMDV rN17 D at the combination property. Thus, the virus rD86 H might have potential in the development of improved vaccines. This study also illustrated that the same amino acid substitution could lead to distinct effects in different serotypes and subtypes of FMDV.