Effects Of Exogenous Flag Inserting On The Molecular Biology And Immunogenicity Of Pig-adapted Foot-and-mouth Disease Virus (Fmdv) O/CHA/90 Strain

Foot and Mouth Disease(FMD)is a highly contagious disease,which affects more than seventycloven-hoofed wild and domestic animals,including cattle,pig and sheep,and is listed as reported disease by World Organisation for Animal Health(OIE).Swine Foot and Mouth Disease(FMD)haspeculiar features in disease control and prevention:the dominant transmission mode is direct contact with animals and virus contamination,seldom via air-borne infection.Serious clinical symptoms are often observed but subclinical infection is rarely seen.Infected pigs shed a multitude of virusesthrough respiratory tract.The virus strain has host-tropism.Sufficient immunogenicity always requires high dose of effective antigen to elicit protective response.Vaccination cannot prevent the spread of swine virus but only delay the outbreak of the disease.Present study of FMDV around the world mainly focuses on the cattle,but not pig.Less is known concerning the prevention and control of FMD in pigs in China.Therefore it is inevitable and urgent to study FMD in pigs so as to better control the severe outbreak of FMD in China.Our study constructs infected clones based on the strain of swine FMDV O/CHA/90.Exogenous labels In VP1G-H ring RGD upstream(-4)and downstream(+10)were inserted.Using molecular biology,immunology and biological information methodology to analyze swine FMDV,we aim to explore the variation tendency of virus pathogenesis,variability,and immunogenicity,and to find vaccine candidate strain who chould differentiate vaccinated animals from naturally infected animals,and to provide theoretical basis for effective control of FMD.1.Exogenous FLAG marker inserted into the upstream(-4)and downstream(+10)RGD of the VP1 G-H loop of swine FMDV O/CHA/90The G-H loop,located onVP1 residues 140-160of structure protein on the caspid surface,was conformationally flexible and the most highly variable region in FMD structural proteins.Swine O/CHA/90 virus was isolated from a pig with clinical signs of infection,belonging to Cathaytopotypeof FMDV serotype O,was widely used for preparation of inactivated FMD vaccine for pigs in Mainland China.In this study,we constructed an infectious clone of swine FMDV Cathay topotype strain o/CHA/90,inserted an eight amino acid FLAG peptide(DYKDDDDK)into the upstream(-4)and downstream(+10)RGD peptide motif of the G-H loop,and successfully rescued the resultant chimeric viruses,vFLAG-O/CHA/90 and vO/CHA/90-FLAG.The chimeric viruseswere found to be similar to their parental virus in characteristics of BHK cell passage stability,CPE,virulence,plaque morphology and replication dyriamics.The tagged virus was also pathogenic to suckling mouse with similar pathogenicity and could be passaged stably in suckling mouse.The results demonstrated the resilience of G-H loop to insertions,tolerance of exogenous gene insertion into RGD motifs,permission of inserting 8 amino acid FLAG into RGD(-4)and RGD(+10)and stable expression of FLAG in passaged labeled virus.2.The FLAG insertion did not interferethe entry of RGD motif to BHK·21 cells viathe recognition of surface integrin receptorThe infection ofFMDV on cells mainly depends on the recognition and binding of host cell surface receptors.The virus assembling and releasing after entry mainly includes three different ways:integrin receptor pathway(RGD motif dependent),heparansulphate HS receptor pathway(RGD motif independent)and uncertain way.Here,BHK-21 cells and CHO cells(CHO-K1?CHO-618?CHO-745 and CHO-677)were infected with the rescued viruses respectively.No plaque formation was observed on CHO cells,but similar plaque morphology was shown on BHK-21 cell.Based on the peptides of G-H loop amino acid 142-157 on the FLAG insertion site,three synthetic F1AGpeptideswere employed in the peptide blocking assay.Our data exhibited that both peptides containing RGD completely inhibited the infection f labeled virus on BHK-21 cells,but the peptide containing KGE lost the inhibition function,which indicated thatthe tagged virus remains the infection function via the integrinreceptor.3.The impact of FLAG epitope insertion on virus immunoreactivity and neutralization reactionTo study the impact of FLAG epitope insertion on virus immunogenicity,infected cell cultures were stained by anti-FLAG and rabbit-anti-FMDV mAbs,and then examined by confocal microscopy.The results indicated positive reaction of virus with both antibodies andtagged virus had good reaction with antibody of FMDV and FLAG.To further test antibody neutralization of the chimeric viruses,convalescent serum from parental O/CHA/90 strain couldneutralize each virus completely,the neutralizing titers(Log10)at 48-74hour were:vFLAG-O/CHA/90 3.08±0.66,vO/CHA/90-FLAG 2.34±0.66,vO/CHA/90 1.77±0.66.But anti-FLAG mAb in mouse could only delay the appearance of virus CPE on BHK-21 cells and the CPE of labeled virusshowed after 48 hours by anti-FLAG mAb reaction,the neutralizing titers(Log10)at 48hour were:vFLAG-O/CHA/902.61±0.05,vO/CHA/90-FLAG 2.07±0.04.We proposed that delayed CPE might be due to either hindering of virus adsorption during virus entry and replication,orchange ofpathogenic characteristics.4.Effect of FLAG insertion position on virus antigenic epitope and FLAG epitope displayAfter passages,the tagged viruses and the parental viruses were sequenced and nucleotide sequences of P1 region were compared.We found that the RGD sequence and the FLAG sequence were stable after passages and the VPI E83K mutations was repeated observed in BHK21 adapted FMDV virus.In the FLAG tagged viruses,three amino acid mutations were found in vFLAG-O/CHA/90:VP3 F54L,VP1 N142S(upstream of RGD of G-H loop),D188G(downstream of RGD).One amino acid mutation was found in vO/CHA/90-FLAG:VP3 M86L.Moreover,3D structure prediction showeddifferent space conformation of the FLAG,G-H loop and RGD motif,and space fold at 141-148 of eight amino acidsequence(FLAG)of vFLAG-O/CHA/90 was fully stretched out and got the full display,while FLAG sequences within 158-165 of v O/CHA/90-FLAG wasbent into a small circle.Furthermore,we produced inactivated vaccine,immuned BALB/c mice(9-10 weeks,20 g)at 0 days,14 days(5 ?g/each time)and detected antibody at 28 days.The result indicated anti-FMDV humoral response was elicited in all the cases,but in only vFLAG-O/CHA/90 vaccinated mouse generated anti-FLAG antibodies.This drastic difference suggested that visible mutationsdid not affect G-H LoopandFLAGdirectly,but may affect the FLAG antigen epitope presentation and chimeric virus antibodies inducing/in vivo through the variation,which changed virus structure and the antigensite.5.Evaluation of tagged virus on pigs pathogenicity and immune responseThree healthy susceptible pig(about40 kg)were infected by mice originated virus with 2×105 LD50 via the neck muscle inoculation respectively.Pigs with parental strains O/CHA/90 performed clinical manifestation significantly,with different degrees of blisters or damagedatrhinoscope,hooffork,coronet and palm area,but no clinical symptoms were presented after tagged virus challenge.FMDV antibody in pigswith tagged strain kept low levels,while FMDV antibody in pigswith parental strain began to rise at3days,reached the peak(Log10=3.15)at 5-6 days and then maintained high levels.Theresults demonstrated that tagged virus did not induce immune responsewell,but the parental strain had proliferation response and could elicit the body's humoral immune response effectively.Furthermore,we produced inactivated vaccine,immuned each 3pigs at 0 days,21 days(10ug/each time/each pig),infected pigs at 28days using 1000ID50O/CHA/90,detected anti-FMDV and anti-FLAG at 21 days and observed continuous 10 days.The results indicated that the labeled vaccine induced higher levels of anti-FMDV at 21 days and 10 days after virus challenge,only marker vaccine vFLAG-O/CHA/90 produced varying degrees of anti-FLAG,which confirmed the experimental results of BALB/c miceimmunization.vFLAG-O/CHA/90 had no pathogenicity on pigs,and produced high level of FMDV antibodiesand positive FLAGantibody after immunization.This coulddistinguish the infected animals fromvaccinated animals,and the virus had the potential candidate as alabeled vaccine strain.Taken together,reverse genetics was employed to construct an infectious clone of swine FMDV Cathay topotype strain o/CHA/90 with insertion of an eight amino acid FLAG peptide into the upstream(-4)and downstream(+10)RGD peptide motif of the G-H loop.We successfully rescuedtagged virus vFLAG-O/CHA/90 andvO/CHA/90-FLAG.The FLAG insertion stably existedin passaged chimeric viruses and sucking mice,with no change of RGD motif on the recognition of BHK-21 cell surface integrin receptor.The marker virus reacted with anti-FMDV and anti-FLAG,and had nopathogenicity on pigsOnlyvFLAG-O/CHA/90 induced antibody response against FLAG in mouse and pig.The three amino acid mutations on structural protein may be beneficial to FLAG epitope display and antibody inducing.The FLAG marker virus not only provided a new way for researcher to study pathogenic mechanism and the immune mechanism of FMDV through tracking FLAG-antigen,but also offered a possiblepreparationfor FLAG marker vaccine in the future,which shows promising way to distinguish natural infected animal from vaccinated animal.