The Influence Of The Foot-and-mouth Disease Virus Leader Protein On The Viral Infection

Foot-and-mouth disease is a highly contagious disease caused by foot-and-mouth disease virus,which mainly infect pigs, cattle and sheep and other cloven-hoofed animals. FMDV is a positive-stranded RNA virus which have only an open reading frame(ORF). The gene of leader protein located at the5’end of ORF. The leader protein plays an important role in the viral virulence. The average length of leader protein is201aa. Currently, national foot-and-mouth disease reference laboratory three Mya98strains FMDV which have distinctive length of leader protein. The length of O/CHN/Mya98/33-P strains FMDV leader is200aa; the length of O/CHN/Mya98/HN1strains FMDV leader protein is201aa; the length of O/GSLX/2010strains FMDV leader protein is202aa.In order to study the influence of three different foot-and-mouth disease virus (FMDV) leader protein for viral infectivity, eight full-length infected cDNA clones were constructed based on O/HN/93strain cDNA using reverse genetic technique which contain the leader protein La,Lb,Lab of Chinese isolates O/CHN/Mya98/33-P and O/GSLX/2010and O/CHN/Mya98/HN1. That eight cDNA plasmids were linearized with Not I, and then transfected into BSR cell which stable express T7RNA polymerase. The BSR-T7/5cell monolayers showed cytopathic effects (CPE) at2days post-transfection. Through RT-PCR analysis can detect the purpose of the chimeric gene sequences, and the sequencing results in line with expectations. Through confocal microscopy observation could detect the non-structural protein of FMDV3A expression. All of this could confirm the successful rescue of chimeric FMDV. The name of virus containing O/CHN/Mya98/33-P strains FMDV leader protein Lab is rOP33Lab. The name of virus containing O/GSLX/2010strains FMDV leader protein Lab is rOGSLXLab. The name of virus containing O/CHN/Mya98/HN1strains FMDV leader protein Lab is rOHN1Lab. The name of virus containing O/CHN/Mya98/33-P strains FMDV leader protein La is rOP33La. The name of virus containing O/GSLX/2010strains FMDV leader protein La is rOGSLXLa. The name of virus containing O/CHN/Mya98/HN1strains FMDV leader protein La is rOHN1La. The name of virus containing O/CHN/Mya98/33-P strains FMDV leader protein Lb is rOP33Lb. The name of virus containing O/CHN/Mya98/HN1strains FMDV leader protein Lb is rOHN1Lb.Pig kidney primary cell infect with eight different strains of chimeric virus, and through real-time fluorescent quantitative PCR detection. We found that the multiplication rate of rOP33Lab is faster than rOGSLXLab and rOHN1Lab. The amount of IFNβ and OAS mRNA induced by rOP33Lab were lower than that other two chimeric virus. The ability of against host transcription of IFNP is almost the same. However the proliferation rate of chimeric virus rOP33La is fastest than chimeric virus rOHN1La and rOGSLXLa. Compare with two chimeric Lb virus, the proliferation rate of chimeric virus rOP33Lb is faster than rOHN1Lb, and the ability of against host transcription IFNβ and OAS mRNA is stronger than rOHN1Lb. These results demonstrated that the leader protein La of O/CHN/Mya98/33-P strains FMDV could enhance the virus replication and the leader protein Lb of O/CHN/Mya98/33-P strains FMDV could strongly inhibit the host IFNβ and OAS mRNA of transcription, which further indicate that the leader protein Lb56G or(and)118R could enhance O/CHN/Mya98/33-P strains FMDV ability to inhibit the host innate immunity.This study lay the foundation for further identification of FMDV leader protein critical sites for inhibiting host innate immunity.