Establishment Of Reverse Genetics System For Classical Swine Fever Virus

Classical swine fever(CSF)is a highly contagious disease caused by Classical swine fever virus(CSFV),which is one of the major infectious diseases that harm the pig industry.Since its discovery,it has been epidemic in many countries in the world,and China is one of the countries that are deeply affected by CSFV.At present,the CSFV strains prevalent in China have undergone major changes in genetic variation,virulence and pathogenicity compared with the previous epidemic strains,which also make the prevention and treatment of CSFV in China face more severer challenges.In order to have better prevention and control of CSF,the pathogenesis and the determinants of virulence of CSFV and other issues still need to be further explored.With the deepening of experimental research and the development of related disciplines,reverse genetics technology has become an important tool of studying RNA viruses,and it plays an increasingly important role in the research of CSFV.By constructing an efficient and economical CSFV reverse genetic system,we can conduct more in-depth research on CSFV at the genetic level and provide more ideas for the traditional research of CSFV.In this study,based on DNA-Launched reverse genetics,an infectious clone containing the full-length genomic sequence of CSFV C strain and virulent Shimen strain was constructed by RNA polymerase II system.By introducing the cytomegalovirus(CMV)promoter at the 5' end of the CSFV genome,and the necessary engineering ribozymes such as hepatitis D virus(HDV)ribozyme and bovine growth hormone(BGH)signal sequence at the 3' end,and then cloning the viral genome into a low-copy bacterial artificial chromosome(BAC)vector,the CSFV genome can correctly replicate translations in host cells and improve the rescue efficiency of the virus.Different from the traditional method of in vitro transcription or cell line construction,the constructed infectious clone in this study can directly transfect PK-15 cells and complete the transcription process in the cells,and then the virus can be directly,rapidly and effectively recovered from cloned cDNA.After successfully recovering the virus of the two strains,the corresponding genetic markers were identified and the biological characteristics were verified.The results of the experiment indicated that the rescued virus has similar biological characteristics as its parental strain,and it has been verified that the introduced genetic marker can effectively distinguish them,and the rescued virus also has good proliferative ability on cells in vitro,therefore it can be used for related reverse genetics research of CSFV.On this basis,we introduced the foreign gene EGFP into the genome of CSFV C strain.Finally,the full-length cDNA clone of recombinant CSFV C strain containing the EGFP gene was successfully constructed.Then we tried to rescue the virus,in order to obtain a recombinant CSFV C strain which can stable passage and expression of EGFP fluorescent label,to visualize the detection of CSFV.However,the fluorescence microscopy showed that the P1 and P2 generations showed weak green fluorescence,but in the subsequent passages,the fluorescence gradually disappeared.It is speculated that due to changes in protein conformation,the normal function of the gene is affected,which leads to EGFP's inability to express and pass in the CSFV genome stably.In summary,this study successfully constructed the infectious clones of the Chinese CSFV C strain and Shimen strain through the reverse genetic system,and the virus can be directly,rapidly and effectively recovered from cloned cDNA.On this basis,an infectious clone of CSFV C strain containing the EGFP gene was constructed.These studies laid the foundation for the further investigation of the pathogenesis of CSFV and the study of CSF marker vaccines.