Comparison Of Effect On Viral Translation Of 5'-UTR Between Chinese Strain And Shimen Strain

Classical swine fever (CSF) virus, the pathogen of CSF, is an economically important and highly contagious disease of pigs, causing serious economic losses to the hog industry. In China, the major way to control CSF is inoculation and the most widely used vaccine is attenuated vaccine. The Chinese strain played an important role in preventing CSF from spreading in China, recognized as a safe and effective vaccine and was widely used abroad.The genome consists of 5'- and 3'-untranslated regions( 5'-UTR and 3'-UTR ) flanking a single large open reading frame (ORF), encoding 12 proteins. 5'UTR contains an internal ribosome entry site (IRES), initiating viral translation by base pairing with ribosome. Chinese strain replicates with a low efficiency in culture cells, which the mechanism is not accurately clear. We speculated that the level of virus titer is related with viral translation level, and translation of CSFV is mainly determined by the IRES of 5'-untranslated region, so we compare the translation level between the Shimen strain and C strain. In this study, plasmid pEGFP-N3-FR, containing the luciferase reporter gene and the IRES of Shimen strain, was replaced by the IRES of C strain. To understand the impact of IRES on virus translation of C strain we compared the luciferase expression level of the two plasmids. We found the luciferase activity between pEGFP-N3-FR and pEGFP-N3-FR-C-5'UTR was not significant, suggesting IRES's little effect on viral translation efficiency.In vitro transcription was still the main reverse genetics system of CSFV, namely the transcription was initiated by a promoter and terminated by a terminator. The product, mRNA, was transfected into cells. mRNA is easily degraded by RNase, which is unconvenient for transfection. In our study, T7 RNA polymerase plasmid was transfected into PK-15 cells, avoiding the operation of in vitro transcription since T7 RNA polymerase specifically binds the T7 promoter and causes efficient transcription of mRNA. In our study, T7 RNA polymerase gene was amplified from BL21 E.coli. by Polymerase Chain Reaction (PCR) and cloned into the downstream of CMV promoter in pEGFP-N3. A vector containing T7 RNA polymerase gene and another vector containing the EGFP gene controlled by T7 promoter were cotransfected into the kidney cell, PK-15. The expression of T7 RNA polymerase was identified by detecting GFP. The results showed that the cell expressing green fluorescent protein was detected, suggesting that the expression of T7 RNA polymerase and the establishment of T7 RNA polymerase expression system were finished, although the cell number expressing GFP is low. We speculated that the low transfection efficiency of PK-15 cell leaded to low efficiency of exogenous gene expression.Plasmid stability included separated stability and structural stability. Because of the impact of external condition, mutation would occur during the process of plasmid passage. It is very important to improve the stability of plasmid in genetic research and production. In this study, plasmid pMC18-CSFV-C viral genome was sequenced and several mutant sites were found indicating the structural instability of plasmid during the process of amplification.It is important to prevent CSF spreading in China by grasping the population dynamics of CSF. In addition, vaccination has played an important role in preventing the spread of CSF in China, but the impact of vaccination on the evolution of csfv is not clear. We collected available genome sequence of CSFV in China and found homologous recombination play a role in viral evolution and genetic diversity. The different evolutionary rate and population dynamics in the vaccine-related group and the nonvaccine-related group indicated that vaccination against CSFV affect the population evolution.