Objective: Study the association of the length of poly(C) tract with virulence of foot-and-mouth disease virus.
Methods: The recombinant plasmids pGEM-XJ/AKT/69 containing the full-length cDNA of FMDV were linearized by Nhe I /Not I and transcribed by T7 RNA polymerase. The in vitro transcripts were transfected into BHK-21 cells using Lipofectamine 2000 reagent. Then, the genetic engineering virus was rescued from BHK-21 cells. The poly(C) tracts were sequenced at different passages of rescued virus, and the pathogenicities were evaluated with 3-day-old mice and BHK-21 cells by detection of LD50 and TCID50.
Results: After six passages in BHK-21 cells, cytopathic effect was observed by microscopy. The rescued virus was rejuvenated once in unweaned mice, and then came back to cell passage. We found that the poly(C) tract of rescued virus was shortened when the virus was passaged twice in BHK-21 cells. Although the data of LD50 in mice and TCID50 in BHK-21 cells showed that the virulence and infectivity of genetic engineering viruses were lower than its parental virus, no significant difference was observed between the genetic engineering viruses with the different length poly(C) tract.
Conclusion: The length of poly(C) tract ranged from 12 to 17 nucleotides did not cause significant influence on the virulence and infectivity of genetic engineering virus.