Detection Of Co-infection Of ALV-B And NDV On DF-1 Cells

The development of animal cell culture technology has made this technology widely used in various research medical fields.The use of animal cell culture to produce enzymes,growth factors,vaccines,and monoclonal antibodies that have important therapeutic value has become an important part of the pharmaceutical biotechnology industry.However,there are always some problems in animal cell culture technology,such as low cell density,unstable serum source,and uncertain composition.At present,many research fields in the world focus on optimizing the cell culture environment,changing cell characteristics,increasing product yield,and ensuring its quality and consistency.Screening the optimum cell culture conditions can effectively increase the efficiency of cell culture,save costs,and promote the perfection of large-scale cell culture methods is extremely important.Therefore,this study optimized culture conditions of DF-1 cells.Avian Leucosis?AL?and Newcastle Disease?ND?will lead to slow growth and development of the body,which will seriously affect the production efficiency.Both of them will bring huge economic losses to the aquaculture industry.In this study,the phenomenon of co-infection of ALV-B and NDV was demonstrated at the cellular level in vitro.It is proposed that ALV-B and NDV have sympathetically promoted replication by analyzing the difference in the level of transcription and infection of ALV-B and NDV co-infected cells.Role,synergistically enhances infection and pathogenicity,and provides an effective reference for the study of joint prevention and control and production of potential dual vaccines.In the process of culturing DF-1 cells,the single factor method was used to select four factors:medium,inoculation density,serum concentration,and double antibody concentration.Through the preliminary experimental results,it was found that various factors affect the cell growth.In terms of culture medium,the effects of DMEM high glucose medium,DMEM low sugar medium,and DMEM/F12 medium were investigated;in terms of inoculation density,the cell concentration of the cell suspension was adjusted to 8 and the effects of different seeding densities were explored;In terms of serum concentration,12 serum concentrations of the growth solution were prepared to explore the effects of different serum concentrations;in terms of double antibodies,7 concentrations of double anti-cell growth solution were prepared and the effects of double antibody concentrations were explored.Repeat experiments were performed based on the initially determined range settings.CCK-8 assay was used to detect cell proliferation.The results of cell growth under the influence of various factors were systematically analyzed and the optimal conditions and scope of each influencing factor were finally determined.The results showed that DF-1 cells grew well in DMEM/F12medium at a seeding density of 5×10⁵ cells/mL-7x10⁵ cells/m L and serum concentrations ranged from 7.5%to 12.5%.The cells were cultured according to the conditions of exploration and used for virus infection experiments.The ALV-B group,the NDV group,the co-infection group,and the PBS control group were independently infected.After inoculation of virus,each group of cells was collected for viral nucleic acid extraction at different time points.Reverse transcription was performed to obtain cDNA.Real-time fluorescence quantitative PCR was used to detect the level of viral transcription.2^-??ctmethod was used for the analysis of relative quantification,while viewing under an inverted microscope.The virus replication and cell activity of DF-1 cells infected with both viruses were determined.The results showed that the peak of transcription in the NDV-infected group occurred at 48h,and the transcription level in the connected group was significantly higher than that in the infected group at 72h.It showed that the two viruses co-infected caused a greater impact on the cells.As the amount of virus increased,the activity of the cells decreased.At the same time,the phenomenon of co-infection between ALV-B and NDV was demonstrated,revealing the ALV-B from unusual perspectives.The cooperative replication and pathogenic effects of NDV not only provide a clear direction for subsequent studies on the cooperative mechanisms of ALV-B and NDV,but also provide a scientific theoretical basis for studies on avian tumour disease or other poultry diseases,and provide new opportunities for the prevention and treatment of poultry diseases.DF-1 cells were cultured in DMEM/F12 medium in a culture density of 5×10 ⁵ cells/mL-7×10 ⁵ cells/m L,and fetal bovine serum concentrations were 7.5%-12.5%,with high culture efficiency,and ALV-B and NDV.Can co-infect the same cell and can promote the transcription and replication of the virus,enhance the virus's ability to infect and have synergy.