Construction Of Type O Foot-and-Mouth Disease Virus Mutants And Analysis Of Their Biological Characteristics

Foot-and-mouth disease?FMD?,of which foot-and-mouth disease virus?FMDV?is the causative agent,is a highly contagious disease of pigs,cattle and sheep,as well as other cloven-hoofed animals.The outbreak of FMD seriously endangers the productivity of livestock and the quality of livestock products,affects economic development,international trade and social stability in the affected areas.Therefore,the disease has been the focus of research by veterinarians worldwide for more than 100 years.FMDV is a picrornavirus and exhibits a high potential for variation under immune pressure,especially its structural protein VP1.Changes of amino acids in VP1 structural protein often affect the stability,pathogenicity,replication capacity and antigenicity of FMDV.Therefore,analysis of the changes of amino acids in VP1 of different FMDVs is of great value for the development of new FMD vaccine.In this study,we analyzed and compared amino acid sequence of VP1 structural protein of classic vaccine strains belonging to different topotype of type O FMDV and different time as well as the current FMDV epidemic strains?SEA-Mya98?.The results showed that the vaccine strains drived from Cathay and ME-SA/Pan-Asia topotype contain some conservative or relatively conservative amino acid in VP1 structural protein.To study the influences of these amino acids for FMDV antigenicity,stability,immunogenicity,replication capacity,etc,we conducted the following research.1.Three recombinant plasmids pQFA,pQFE and pQFC were constructed by introd uced three?VP1:H28Q+S47Q+V194I?,six?VP1:H28Q+S47Q+S58A+V194I+A198Q+S212L?and seven?VP1:H28Q+S47Q+S58A+V194I+S197D+A198Q+S212L?amino aci d substitutions into the chimeric full-length clones containing the VP1 gene of the c irculating FMDV.The genetically engineered FMDVs were successfully obtained after transfection the linearized recombinant plasmid into BSR/T7 cells.The recombinant FMDVs were confirmed by RT-PCR,nucleotide sequence analysis,indirect immun ofluorescence and electron microscopy.The results showed that we successfully rescu ed three FMDVs containing the target mutation,indicating the substitutions?three,si x and seven amino acids?in FMDV VP1 did not affect FMDV viability.2.The genetically engineered FMDVs have similar plaques sizes and growth kinetics with the parental virus,but thermal stability after treatment at 37?for 3h,6h and 9h and at 42?for 0.5h and 1h is lower than that of parental virus.The LD50 values of milk mice enhanced 4 times,demonstrating that the substitutions?three,six and seven amino acids?in VP1 structural protein of FMDV do not significantly affect the virus plaques phenotype and the replication ability,but affect the thermal stability and the pathogenicity for milk mice.3.The cell-adapted FMDVs O/HN-7/2010?SEA-Mya98?,O/HK/CHA/99?ME-SA/PanAsia?and O/GX/CHA/2009?Cathay?,which are non-pathogenic in C57BL/6adult mice,were passaged 4 times in the suckling mice.The O/GX/CHA/2009MF4 and O/HN-7/2010MF4 which are sensitive to C57BL/6 mice were obtained and the LD₅₀0 was10^4.2 and 10^4.5 respectively.The FMDV O/HK/CHA/99MF4,still is non-pathogenic in C57BL/6 adult mice,was subjected to in vivo?C57BL/6 mice?and in vitro(Fetal pig kidney cells?FPK?cycle passages for nine times.The C57BL/6 mice appeared clinical signs and death when five cycles replication were completed.With the increase of passage times,the pathogenicity for C57BL/6 mice was increased.LD₅₀0 values of the fifth and ninth passage were 10^2.8and 10⁵.1.1 respectively.These FMDV strains,which were pathogenic for C57BL/6 adult mice,would lay a basis for the pathogenicity and vaccine efficacy evaluation in C57BL/6 mice in the future.4.The genetically engineered viruses?r-FMDV/MyVP1-A?r-FMDV/MyVP1-C?r-FMDV/MyVP1-E?and parental virus were propagated in BHK-21 monolayers.The chemically inactivated FMD vaccines were preparated and immunized guinea pigs with different doses?1?g and 2?g of 146S antigen?respectively.After 14,21,28 and 35 days postvaccination,sera samples were collected from all animals and neutralizing antibodies against FMDV were tested by serum neutralization assay.The results showed that the level of neutralizing antibodies gradually increases with the extension of time and it reaches the peak at 28d or 35d postvaccination.The titers of r-FMDV/MyVP1-A?r-FMDV/MyVP1-C vaccine-induced neutralizing antibody against corresponding viruses were obvious higher than the titers of parental vaccine-induced neutralizing antibody against parental virus?P<0.05?.The neutralizing antibody levels induced by vaccines made from three genetically engineered FMDVs were significantly lower than that of parental virus?P<0.05?.Neutralizing antibody titers against FMDV O/HN-7/2010,O/HK/CHA/99 and O/GX/CHA/2009 were tested by a VNT,and the vaccine matching relationship?r?values were calculated.The data showed that the r value between genetically engineered FMDVs and the current popular FMDV strains were obvious lower than that of parental virus.All the results demonstrated that the substitutions?three,six and seven amino acids?in VP1structural protein of FMDV alienated the antigen relationship between the genetic engineering FMDV and its parental virus and all genetically engineered FMDVs were not a good match with the circulating virues campared with the parental virus,while the substitutions?three,seven amino acids?significantly enhance the immunogenicity of genetically engineered FMDVs.5.8-week-old C57BL/6 mice were inoculated intramuscularly with four experimental vaccines,respectively.After 7 days post-vaccination,serum samples were collected and all mice were challenged with 100 LD₅₀ of O/HN-7/2010,O/HK/CHA/99 and O/GX/CHA/2009.The results showed that all C57BL/6 mice inoculated with the vaccines were fully protected against the FMDV challenge,it is speculated that high potency vaccines?0.8?g?caused completely protection.Our results were consistent with previously reports that high potency FMD vaccine in animals can resist heterologous challenge.Additionally,all the mice inoculated with experimental vaccines obtained high antibody levels?1:32?,while the antibody levels induced by the parental virus were significantly higher than that of the genetic engineering viruses,which is consistent with the result of guinea pigs.This study firstly reported that the substitutions of 6 and 7 amino acids introduced into FMDV structural protein VP1 exert influence on FMDV stability,pathogenicity,replication capacity,immunogenicity etc.,which will lay theoretical basis for the development of excellent FMD vaccine candidate strains in the future.