Construction Of Infectious Genomic CDNA Clones For CSFV Shimen Strain And Researches On Defective Virus-induced Cytopathic Effect

A high death rate will occur for pigs infected by classical swine fever virus (CSFV) virulent strain. Great loss for worldwide development and trade of stock raising has been generated from prevalence of classical swine fever, especially of recent nontypical classical swine fever (CSF). No cytopathic effect (CPE) can be observed for CSFV when cultured in vitro on host cells though serious damages occur in many tissues and organs for in vivo infection in pigs. Researches on pathogenesis of CSFV are always delayed for lack of suitable in vitro cytopathic cell model.Recently three cytopathic defective interfering (DI) particles of CSFV were isolated from the nature outside of China. Investigations based on reported DI particles uncovered presence of subgenomic RNA was responsible for induction of CPE in host cells. Nonstructural proteins NS2 and NS3 are present as diad NS2-3 in wildtype noncytopathic CSFV. NS2 gene is deleted together with all upstream coding regions in CSFV DI particles, and diad NS2-3 is changed to NS3 monomer. Furthermore, over expression of nonstructural protein NS3 in cell culture of cytopathic CSFV was considered as hallmark of DI particle-induced CPE.Full genomic sequence of CSFV persistent infection strain CSFV39, which resulted from nontypical CSF, was determined firstly in this dissertation. Full genomic and proteomic sequence alignments of CSFV39 between Chinese standard virulent strain Shimen and vaccine strain HCLV suggested a higher homology between CSFV39 and Shimen strain. Generation of persistent infection was speculated from virulence attenuation of virulent CSFV under immunity pressures during chronic prevalence. Under this situation, step researches on pathogenesis of CSFV virulent strain become challenges for present field of basic theory. We selected CSFV virulent Shimen strain as research material, then constructed a platform of CSFV genomic infectious cDNA clone based on its reported complete sequence. This cDNA platform was then employed to the generation of cytopathic cell models of CSFV for in vitro researches. Based on one of the models, molecular mechanism for CPE induced by subgenomic CSFV virus was studied.The full-length genomic cDNA clone pT7SM of CSFV was obtained by RT-PCR amplifications and gradual ligations between overlapping genomic cDNA fragments. Full genomic CSFV vT7SM was rescued in vivo by transfection of linear plasmid pT7SM/SacII into PK-15 cells with induction of T7 RNA polymerase expressed by mutant vaccinia virus. Two subgenomic cDNA clones of CSFV were successively constructed based on above infectious pT7SM with gene deletions, then applied to establish two cytopathic cell models for in vitro researches of CSFV. Construction of the model depending on subgenomic CSFV virus vSMD2 aimed to confirm the report that the natural subgenomic DI particles induce CPE on host cells, while the other defective CSFV vSMD8, based on deletion of nonstructural protein NS2, were designed for researches on molecular mechanism for CPE induced by subgenomic virus. Apoptosis induced by vSMD8 was the most interesting find with this cytopathic cell model.Functions of NS2 were then explored during CPE induced by defective vSMD8 with above cytopathic cell model. A cell line PK-15/NS2 expressed CSFV NS2 protein was screened with G418 on PK-15 cells transfected with eukaryotic expression plasmid of NS2 gene. CPE was dramatically inhibited on PK-15/NS2 infected by defective virus vSMD8. The result demonstrated the complement function of NS2 protein in PK-15/NS2 cells for deletion of NS2 gene in the defective subgenome of vSMD8 used for infection. It was suggested that NS2 protein could inhibit the ability of CPE induction for over expression of CSFV nonstructural protein NS3 in host cells, whether in style of individually expressed monomer in PK-15/NS2 or diad with NS3 (NS2-3) that presents in cell culture of wildtype CSFV.