Construction And Comparative Analysis Of Biological And Immunoserological Properties Of Site-directed Mutants Containing Amino Acid Mutations In VP3 Of Foot-and-Mouth Disease Virus

Foot-and-mouth disease（FMD）is one of the serious animal diseases that affects cloven-hoofed animals worldwide and causes severe economic losses due to the decline of livestock production and international trade restrictions on animals and animal products.The causative agent is foot-and-mouth disease virus（FMDV）that exists as three of seven immunologically distinct serotypes（O,A,and Asia 1）in China.Serotype O is one of the most prevalent and widely distributed serotypes of FMDV,which is currently considered the most acutely threatening form of livestock breeding and cultivation.Vaccination is one of the significant preventative measures and there are two primary types of commercially available vaccines for the control of FMD:conventional inactivated vaccine and synthetic peptide vaccine.Although effective,crucial and wildly used for the preventing clinical signs of the disease,the conventional FMDV inactivated vaccine consisting of whole virus antigen could also contains non-structural proteins（NSPs）due to poor purification procedures.When animals have been vaccinated repeatedly,the inactivated vaccine will interfere with the differentiating of infected from vaccinated animals（DIVA）.Synthetic peptide-based vaccine does not exhibit antibody reactivity of FMDV NSPs,however,the vaccine is limited by its narrow antigenic spectrum and poor cross protection.With the progress of reverse vaccinology,a negative-marker vaccine against FMDV has been generated by using genetic engineer ing techniques,for a DIVA matching test based on detecting FMDV NSPs antibody.In the present study,the foreign tags,the indicated inter-serotypic regions or site-directed mutations were introduced into the G–H loop of the surface-exposed capsid protein VP3 of FMDV for the recovery of expectant recombinant viruses.Our results may help to further development of broad antigenic spectrum,polyvalent and combined,or positive-marker FMD vaccines.To evaluate the capability of expressing foreign tags in the G–H loop of the surface-exposed capsid protein VP3,HA tag was inserted into 3171–3172 residues for the production of two FMDV genome-modified constructs,by using an infectious c DNA（pOFS）of Cathay virus（r HN）as the original backbone.Unfortunately,it was not possible to generate infectious progeny viruses following transfection with these two linear ized plasmid c DNAs and successive blind passages of the corresponding transfected supernatants in cultured cells.Then,HA,Flag,VSV-G tags were independently substituted into different positions of the G–H loop of VP3,to construct six expectant recombinant full-length genomic clones.There were still none of infectious progeny viruses rescued successfully after transfection of the corresponding mutated plasmids.Synamous codon usage in the plasmid-der ived region of one of the HA-substituted c DNAs did not give rise to our desired outcome.These results suggest that some specific amino acid residues in the G–H loop of VP3 of r HN are essential for maintaining viral infectivity.To identify the functional amino acid residues（s）in the G–H loop of VP3 critical for the infectivity of r HN,four site-directed mutated full-length genomic c DNAs（comparable to that of the indicated HA-substitued c DNA）and two inter-serotypic chimeric c DNAs（as compared to C3 Resende/1/55 and SAT2/ZIM/7/83）were produced by site-directed mutagenesis.The results of transfection showed that A3168P could exert a deleter ious effect for the propagation of infectious viruses.Nevertheless,two site-directed mutants,r HN^V3174Y and r HN^{D3173N+V3174E+N3179C},were rescued from the indicated plasmid derivatives using reverse genetics.Subsequently,the potential role of site-directed mutation（s）in the G–H loop of VP3 in response to phenotypic properties and immunoserology of r HN,r HN^V3174Y and r HN^{D3173N+V3174E+N3179C},were characterized in duplicate or more.And the results from series assays demonstrate that（i）pseudoreversions and second-site mutations occurred in the capsid coding region of r HN^{D3173N+V3174E+N3179C} during serial passages in BHK-21 cells;（ii）r HN^V3174Y and the passaged variant and genetically stable derivatives of r HN^{D3173N+V3174E+N3179C} gained the RGD-independent ability to infect CHO cell lines;（iii）r HN^V3174Y and the genetically stable virus of r HN^{D3173N+V3174E+N3179C} showed a somewhat moderate change but no significance in plaque size and replication dynamics in the infection of BHK-21 cells compared to r HN.As compared with that of r HN,r HN^V3174Y and the genetically stable virus of r HN^{D3173N+V3174E+N3179C} displayed a lower level of the virulent phenotype.Relative to r HN^V3174Y,it was noticeable that D3174N and N3179C of the genetically stable clone of r HN^{D3173N+V3174E+N3179C} could result in an additive effect on viral pathogenicity in vitro and in vivo.Furthermore,the antibody cross-protection（such as Mya98 and PanAsia-1 strains）of two site-directed mutants,especially the genetically stable virus of r HN^{D3173N+V3174E+N3179C} were more effective to neutralize PanAsia-1 lineage of FMDV serotype O（OIE Standards Complied,r value≥0.3）.According to the above results,it seems that the introduction of amino acid mutations in non-B cell epitope domains,in close proximity to the antigenic sites of the mature FMDV virion（such as VP3 G–H loop）,could improve the immunological efficiency of FMD vaccine candidates,in the future.