A Study Of Proliferation And Induce Cells Apoptosis Characteristics Of Different Virulence Of Aleutian Mink Disease Virus In CRFK Cells

Aleutian disease of mink(AMD)is one of the major diseases that endanger the world's mink industry.The pathogen of Aleutian mink disease virus(AMDV)infection is chronic,persistent and progressive infection.The quality of the fur and the ability of the reproductive capacity were decreased,also known as plasma cellosis.Due to the disease pathogenesis of is special,there was no a valid vaccine for the prevention and treatment of the disease.Studies have shown that low levels of replication and expression of AMDV can lead to persistent infection and immune complex formation of the mink,causing chronic diseases,and the apoptosis of cells plays an important role in this pathogenesis.In order to obtain the most common method of attenuated vaccine candidate strains we often study the pathogenesis of the virus and virulent weak in vitro culture of the virus.In this study,we cultured the virulent strains which isolated and identified in the laboratory in vitro.The proliferation characteristics and apoptosis characteristics of different virulent and different virulent viruses were studied.Which can lay the foundation for The pathogenesis of AMDV and the attenuated vaccine Strain screening.The specific PCR primers were designed and the reaction conditions being optimized to the Fluorescence Quantitative PCR Assay,The method was simple,specific and sensitive.Which provide the technical support for AMDV clinical detection and AMDV evaluation of proliferation of CRFK cells.AMDV-DL124 and AMDV-DL125 of virus of generations 7(F7)were cultured in CRFK cells in vitro.The culture conditions were optimized and cultured to generations 45(F45).The proliferative characteristics of AMDV-DL124?AMDDV-DL125 strain and AMDV-G strain were detected by TCID50,virus immunoassay and fluorescence quantitative PCR.The results showed that the viral content of AMDV-DL124 and AMDV-DL125 strain was up to 108.5 TCID50 / mL,which was significantly higher than that of 106 TCID50/mL of F45 and the105.5 TCID50/mL of AMDV-G.At the same time of the viral content of F45 and the AMDV-G was no significant difference.The results of indirect immunofluorescence showed that the proliferation rate of AMDV-DL124 and AMDV-DL125 strains was faster at 48-72 h,and the proliferation rate was slower at 12-36 h.The results of the fluorescence quantification of PCR shows that the number of AMDV-DL124 and AMDV-DL125 nucleotides was up to 2.4 × 106,which was significantly higher than that of F45 and the number of AMDV-DL124 and AMDV-DL125 nucleotides was significantly higher than that of F45 Virus and AMDV-G strain virus,and then no significant difference between the two.According to the The results showed that the replication of AMDV-DL124 and AMDV-DL125 were significantly reduced in CRFK cells after CRFK cell culture.The apoptosis of CRFK cells induced by AMDV-DL124,AMDV-DL125 strain F7 and F45 viruses and standard attenuated strain AMDV-G was detected by TUNEL method and flow cytometry.The results of TUNEL assay showed that the apoptotic rate of CRFK cells induced by AMDV-DL124 and AMDV-DL125 strain showed that the poptosis of virus was observed at 12 h after infection,and the apoptotic of AMDV-G were observed at 24 h after infection.the apoptotic of AMDV was detected by flow cytometry showed that the apoptotic rate of CRFK cells induced by AMDV-DL124 and AMDV-DL125 strain was up to 15.86%which higher than that the apoptotic rate of F45 and AMDV-G strains was 2.94% and 1.65%.hese results indicate that the apoptotic rate was focus on 2 ~ 12 h after virus infection.The ability of apoptosis of AMDV-DL124 and AMDV-DL125 strains to induce apoptosis after continuous passage in CRFK cells is reduced.This study laid the foundation for the study of the relationship between pathogenesis of AMDV and the apoptosis of cells.