The Influence In Replication Of CSFV After The Gene Of CSFV NS2 Transfected Into The PK-15 Cells

Classical swine fever (CSF) is a highly contagious swine disease with high morbility and mortality, featuring symptoms of hemorrhagic fever and immune suppression. Classical swine fever virus (CSFV) is the pathogen of CSF. CSFV is a small, enveloped virus and the diameter of the CSFV partical is 40 nm-50 nm. The virus belong to the genus Pestivirus within the family Flaviviridae and the genome is a single-stranded RNA of positive polarity encoding one large polyprotein which is further processed into mature proteins. The viral genome is approximately 12.3 kb in size and contains a single large open reading frame (ORF). This ORF is translated into a polyprotein which is further cleaved into 12 mature proteins:Npro, C, Erns, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B. The disease is a notifiable one of the World organization for animal health (OIE), usually leading to significant economic losses to the pig industry in many countries. In recent years, CSF with lower morbility and mortality and mild, chronic, atypical symptoms was often observed, even in immune pigs herd. People must pay attention to the new problems and find some new methods to confront the difficulties as soon as we possibly can. Even more daunting is the task to globally monitor the impact of viral infection on the genome of the host cell and many technical hurdles must still be overcome in order to develop new pharmaceutical product for prevention and cure CSF.The key of the new methods is the study of CSFV pathogenic mechanism deeply. Viruses constantly adapt to and modulate the host environment during replication and propagation. At present, a little is known about the interaction between CSFV and the host cells. The function of NS2 gene is known less than the function of other non-structural region gene. But NS2 gene plays an important role in CSFV replication while CSFV is infecting cells. So, the study on pathogenic mechanism was carried out, the gene of CSFV NS2 was transfected into the PK-15 cells. Then CSFV infect the PK-15 cells.Firstly, a pair of specific primers were designed according to strain AF092448 published on Genbank to amplify CSFV NS2 gene by PCR from the viral genome. We insert restriction enzyme, his tag, start codon and termination codon into the primers, and cloned them into retrovirus expression vector pLXRN after sequencing correctly. The recombinant vectors were named pLXRN-NS2. The recombinant plasmids were transfected into porcine kidney cells PK-15. G418-resistant PK-15 clones were obtained under the antibiotics pressure. With PCR analysis we were sure the gene of NS2 was integrated into the genome of PK-15. To uncover the influence in replication of CSFV, we examined the CSFV genome duplication by using real-time quantitative polymerase chain reaction (PCR) after CSFV infection. Real-time PCR is a sensitivity, specificity and rapid method for CSFV detection. In this study, we have successfully used a TaqMan real-time PCR system that can detect the content of CSFV in the cells. We extracted RNA from the PK-15 cells which infected CSFV in 24,48 and 72 hours and used real-time PCR to detect the content of CSFV. The content of CSFV in the normal PK-15 cells with the content in the PK-15 cells was compared after the gene of NS2 transfected, the result show that the content of CSFV in the normal PK-15 cells is more than the other one. And the difference is predominance. After being infected, the content of CSFV in the cells without transfection is 13.6,163.6 and 104.4 times of the content with transfection at 24,48 and 72 hours respectively. So we obtain the conclusion that NS2 gene inhibits CSFV replication after transfection into the PK-15 cells. The study settles the base of the interaction between NS2 gene and replication of CSFV and promotes the research of pathogenic mechanism of CSFV. The study provides a new direction for developing new pharmaceutical product for prevention and cure CSF.To sum up, NS2 play an important role in CSFV replication. Current studies suggest that the incidence and development of virus disease is a multi-gene, multi-step, multi-stage process of participation, in which CSFV NS2 gene need to be further studied.