Biological Characterization Of Natural Recombinant Virus Of Asia 1 Foot-and-Mouth Disease Vaccine Strain

Foot and mouth disease(FMD)is listed as a legally reported animal disease by OIE,which is also one important diseases concerned by international trade and has a huge influence on economy and politics.Based on the national conditonals and the epidemic situation,different countries and regions adopted culling or combined culling with vaccine immunization to prevent and control foot-and-mouth disease effectively.The quality of vaccine product is the most important index of vaccination.Therefore,the good quality of the vaccine product,to provide material support for prevention and control is also crucial.In this study,the abnormalities occurred in the cultivation and quality inspection of Asian type 1vaccine against foot-and-mouth disease were studied,aiming to provide new ideas for the monitoring and quality control of Asian type 1 vaccine.In this study,we found that the foot-and-mouth disease virus Asia-1/HN/06 with a 12nt insertion(GTTGACTTAGTA,encoding VDLV)at the 3'terminal of VP1 gene(following the 619th nucleotide)during passage in serum-free suspension culture of BHK-21 cells.Next generation sequencing confirmed that the virus presented insert mutation in the 4th generation of serum-free suspension culture BHK-21 cells,and the proportion of the virus variation increased with the increase of the passage.The mutant virus strain containing 12nt insertion was isolated by plaque cloning and named VAC-Asia-1-VDLV.In order to determine the origin of the 12nt insertion sequence,the nucletide sequence was analyzed by Blast and transcriptome sequencing was performed on the host cells.It was confirmed that the 12nt insertion sequence was derived from BHK-21 cell mRNA.Through analysis of the sample reads database,three mRNAs in the BHK-21 cell transcriptome contain 12 nt identical sequences.Combining with matching flanking sequence length,RNA recombination mechanism and the change of mRNA level analysis,it was found that the expression level of secernin-3 protein is significantly higher.In the control group,and the mRNA contains the longest matching sequence with 12 nt and two wings,it is the most likely to be recombined as a donor and virus.The results of biological characteristics showed that VAC-Asia-1-VDLV virus showed no obvious morphological changes of plaque in BHK-21 cells,but could grow in CHO-K1 derivative cells,and the titer of the virus exceeded 1×10~5PFU/mL,while the infection rate of Asia-1/HN/06 in CHO series cells was very low and there was no plaque.It is indicate that the 12nt insertion virus,VAC-Asia-1-VDLV,can infected cells with integrin and HS receptors deficience through novle receptors.The 12nt nucletide sequence encoding VDLV was inserted into the O/CHA/90 virus by reverse genetic operation,and the O/CHA/90-VDLV infectious molecular clone was constructed.The results showed that the plaque morphology,growth curve and infection to CHO-K1 series cells compared the virus to the parent strains O/CHA/90 had no obvious change,indicating that the 12nt inserted into the virus has a little influence on the growth characteristics in BHK-21 cells,and has a limited effect on the use of the novel receptors.The results showed that VAC-Asia-1-VDLV and Asia-1/HN/06 virus had no significant difference in pathogenicity of suckling mouse,but the pathogenicity of cattle was quite different.None of the 3cattle inoculated with VAC-Asia-1-VDLV virus showed clinical symptoms or body temperature response or obvious viremia.Cattle inoculated with Asi-1/HN/06 virus showed typical symptoms of foot-and-mouth disease and body temperature response and obviouse viremia.The results showed that the virulence of VAC-Asia-1-VDLV was significantly attenuated in cattle,and the reason was that the mutation of RGD into GGD and 12nt insertion sequence significantly affected the virulence of Asia-1foot-and-mouth disease virus in cattle.This is the first report showing recombination between a FMDV genome and genomic information from cells in which it has replicated.The source of donor mRNA was analyzed by transcriptome sequencing,and the effects of recombinant virus receptor utilization and pathogenicity on the animal were studied.In this study,the variation of the adaptor cells of Asia 1 foot-and-mouth disease virus has a great influence on the immune effect,and it also provides sufficient evidence for the necessity of monitoring the virus in the production of foot-and-mouth disease vaccine.