Construction Of The CDNA Clone And Chimeric Clones Of Classical Swine Fever Virus C-strain Vaccine

Classical swine fever (CSF), which is caused by classical swine fever virus (CSFV), is a highly contagious and often fatal viral disease of pigs. It is characterized by high fever and hemorrhages, and classified as an OIE list A disease. Great losses to pig industry worldwide have been caused by the prevalence of CSF, especially of nontypical forms. Hog cholera lapinized virus (HCLV) (also known as C-strain), a very safe and efficacious vaccine developed in China, has been playing a key role in the control of the disease. However pigs vaccinated by this vaccine cannot be distinguished from wild-type CSFV infected pigs by conventional serological assays. Development of marker vaccines may help to solve this problem.This study aims to construct the full-length cDNA clone of HCLV to establish a reverse genetics system as platform for study of CSFV. Nine primer pairs were designed according to the published sequences of HCLV. Using RT-PCR, nine overlapping cDNA fragments (F1-9) covering the complete genome of HCLV/HVRI were amplified, cloned and sequenced, and then the complete genome sequence was assembled. Homology comparison between the HCLV/HVRI sequence and other 7 HCLV sequences shows that the HCLV genome is rather genetically stable. Using one-step ligation of multi-fragment method, two half-length cDNA clones pOK12/F1-5 and pOK12/F6-9 were obtained by cloning fragments F1 to 5 and F6 to 9 into pOK12. Then the two half-length cDNA clones were ligated, resulting in the full-length cDNA clone pOK12/F1-9 (pOKHCLV). By insertion, substitution, deletion in pOKHCLV, we obtained a series of chimeric clones pOKHCLV/EGFP (inserting EGFP gene into HCLV genome), pOKHCLV/EIAVep (inserting an EIAV epitope into HCLV genome), pOKHCLV/3'UTR (substituting 3'-untranslating region of Shimen for that of HCLV), pOKHCLV/Shimen (substituting 3'-half part of Shimen strain for that of HCLV), pOKShimen/HCLV (substituting 5'-half part of Shimen strain for that of HCLV), pOKHCLVdel4764 (deleting 4764 bp in the coding region of HCLV), and pOKCIHCLV (placing HCLV genome under the control of CMV promoter). The molecular clone and chimeric clones can be used to study the virulence determinants of HCLV and develop marker vaccines against CSFV.