Establishment Of In Situ RT-PCR To Detect Classical Swine Fever Virus And Study On Molecular Pathogenesis Mechanism Of Classical Swine Virus

According to complete gene sequence of Classical Swine Fever Virus (CSFV) strain of Shimen, two pairs of primes, PC1, PC2, PC3 and PC4, were designed, and the highly conserved gene region (gene C) was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) with these primes. Gene C was cloned into pGEM- T easy vector. The cloned vector was amplified by DH5α.After digested by EcoR Ⅰ and Spe, the templte DNA was done. A probe was prepared by labeling Digoxigen with random primed, and the sensitivity of probe was 1pg. A new in situ RT-PCR method was developed in this study .Shimen strain (group A) and Chinese strain (group B) of CSFV was localized in some tissues by in situ RT-PCR. Shimen strain of CSFV already infected in tonsils, spleen, kidney, lymphaden nodes, liver since 4d post infection (PI) by in situ RT-PCR. From PI 7d to death virus began replicating in these tissues and induced seriously damage. While Chinese strain of CSFV (group B) first infected tonsils, spleen, kidney, lymphaden nodes on PI 2d, then infected liver on PI 4d, finally infected kidney on PI 7d. No virus could be deteted in these tissues on PI 20d. From PI 2d to PI 16d,both CSFV of brood taken in group A and Group B could be detected by nest RT-PCR.but in group A nothing could be detected on PI 20d.In this study virus could be detected in both group A and group B. in which pigs were inoculated with Chinese stain and Shimen strain,respectively.The results of in situ RT-PCR in group A has 66.7% identity with rabbits test. The results of group B by in situ RT-PCR has 88% and 84% identity with cryostat sections and plaster by direct fluorescence assay. The identity between cryostat sections and plaster by direct fluorescence assay was 80%.During the experiment a series of blood samples were taken from three groups and 16 hematological valus and 15 biochemical values in serum were determined, then analyzed their sequential changes. ALB in serum biochemical values was significant differences between group A and group B (P<0.05). Being short of WBC, GR, LY, there were no other significant differences values in hematological in two groups. BUN in serum biochemical values was significant differences between group B and group C (P<0.05), but GR%, PLT, PCT values were significant differences in hematological in two groups (P<0.05). TB1L in serum biochemical values was significant differences between group A and group C (P<0.05), but only PCT was significant differences in hematological values (PO.05).The results implied that In situ RT-PCR technique developed in this study was useful for detection of CSFV RNA in tissues, and maybe a valuable technique for studying the pathogenesis of persistent infection of CSFV. In this study, 16 valus in hematological and 15 values in serum biochemical of blood were first analyzed among three groups, and were used to study their pathogenesis on pathologic physiology .