The Relationship Of Structural Protein Of CSFV With Biological Characteristics

Classical swine fever (CSF) caused by the classical swine fever virus (CSFV) is a highly fatal and contagious viral disease of swine in the world. It is a must reportable disease in the list of the Office International des Epizooties (OIE). Classical swine fever virus, SM (shimen) strain, is a virulent strain and could not induce rabbit fever as a reference strain in China. Hog Cholera Lapinized Virus (HCLV, or C strain), attenuated strain from virulent strain in rabbit, shows poor growth in cells culture, but it causes a typical fever reaction in rabbit. Thiverval strain (T strain) is a temperature-sensitive mutant strain from Alfort strain and appeares good growth in cells culture without fever reaction in rabbit. In this study, infectious clone of T strain the structural protein coding region was replaced with the same region of C strain and SM strain respectively. The chimeric viruses, vCT and vSMT, were rescued successfully from infectious clone pCT and pSMT using reverse genetic techniques. The chimeric viruses were evaluated in cell culture and rabbit to identify the relationship of structural protein of CSFV with growth characteristic in cells culture and rabbit fever reaction.By comparing the full-length genome of CSFV Cstrains, SM strains and T strains published in NCBI, three pairs of primers were designed and synthesized. The complete structural protein-coding region of CSFV C strain and SM strain had been amplified by RT-PCR.On the basic of the infectious cDNA clone of CSFV T strain, complete structural protein-coding region of T strain had been replaced by C and SM strain respectively. Two chimeric plasmids had been constructed successfully and identified by two sets of restriction enzyme. Genomic RNA was transcribed in vitro and further transfected into BHK-21 or SK6 cell line by electroporation. Two chimeric viruses had been rescued. The appropriate conditions for electroporation transfection SK6 were been established. The rescued viruses were passaged on SK6 cells using freezing-thawing virus infected cells. The viruses also were collected from supernatant of split virus-carrying SK6 cell. The virus was identified using immunohistochemistry stain with CSFV E2 McAb WH303. Amount of virus were determined by PFU and ELISA. The chimeric viruses were confirmed by RT-PCR and sequencing. The result showed that higher titer of virus could be obtained from supernatant of split virus-carrying SK6 cell. The titer of chimeric viruses was lower than that of T strain. It was concluded that the structural protein of C strain and SM strain effected growth in cells culture to some degree.To investigate the interaction structural protein of the CSFV with rabbit fever, the rabbits were divided into five groups. In group1,2 rabbits were intravenously inoculated with 50RID C strain; group2,2 rabbits with 2ml mock medium; group3,5 rabbits with 107PFU T strain; group4,3 rabbits with 1.8×103 PFU chimeric virus vCTFO; group5,3 rabbits with 103 PFU chimeric virus vCTF1, respectively. The results showed that a certain number of chimeric virus vCT could, induce rabbits fever reaction as C strain did, and the T strain could not make rabbits fever. So we found that structural protein of CSFV strain C was responsible for the rabbit fever reaction.To confirm the propagation of chimeric viruse vCT in rabbit, the blood was collected and organs samples of spleen and lymph node were obtained and fixed in 4% paraformal-dehyde and processed for paraffin embedding for histopathological studies. The distribution and viral loads of CSFV were detected by immunohistochemistry(IHC). CSFV-specific blocking ELISA kit was used for the detection of Erns in serum and tissue. Clinical symptoms and necropsy changes were recorded. Spleen was bleeding, erythrodegeneration, some granular cell appeared in part of red pulp, the area of white pulp decreased obviously, lymphocyte increased, lymphoma were soakaged by lots of macrolymphocyte. What is more, positive substance distributed throughout medulla of lymphoma and red pulp of spleen on the whole, and the positive cell included mainly reticular cell and macrolymphocyte. These indicated that chimeric viruse vCT had multiplicatied in rabbit and led to tissue damage and immune response of body.To sum up the data above, the structural protein of CSFV strain C and strain SM was involved in growth characteristics in cells culture. The structural protein of CSFV strain C influenced rabbit fever and could transfer to T strain chimeric virus vCT. All of these pave a way to develop growth ability in cell culture and modify product method of CSFV strain C, and lay the foundation for further research for DIVA(Differentiating Infected from Vaccinated Animals, DIVA) vaccine.