Establishment Of ALV-Jin Vitro Infection Of PBMC

Avian leukosis is a general term for a variety of avian tumor diseases caused by viruses of the retrovirus A genus,which,since discovered,has spread among chickens over many countries.It usually arouses leukemia diseases such as lymphocytic leukemia,myeloblastic leukemia,myeloid leukemia,etc.,and immunosuppression,serving one of the threatening diseases for poultry industry,and among which the Subgroup J avian leukemia is one of the most harmful leukemia diseases in clinic.However,current researches of ALV-J are mainly focused on epidemiology,pathogenicity,molecular structure and function,etc.,but few research concerns virus and host immune response.In particular,it remains unclear how the host produces antiviral responses to ALV-J.In addition,former researches mainly focused on single cell lines ignoring the fact that,PBMC,as a mature lymphocyte population in the peripheral blood circulation consists multiple immune cells such as T lymphocytes,B lymphocytes,NK cells,and mononuclear macrophages,for which PBMC serves as a research sample of avian leukemia virus（ALV）,infectious bursal disease（IBDV）,Marek’s disease virus（MDV）and other immunosuppressive viruses that are harmful to the poultry industry.In order to clarify how ALV-J antigen specific memory PBMC produces antiviral cellular immune response to ALV-J,this study attempts to explore the changes in immune response caused by ALV-J in vitro.to study the subsequent host immune response and interaction,we construct the model of the memory PBMC infected by subgroup J avian leukosis virus（SCAU-HN06 strain）in vitro.The model considers the changes in the total number of live cells and the PBMC infection status,and chooses the virus titer with the highest relative virus copy number and the highest S/P value caused by the virus infection of memory PBMC(10^4.5TCID₅₀)as the virus titter for subsequent in vitro infection experiments;it also chooses 4 HPI（Hour post-infection,HPI）and 8 HPI as the detection time points for immune cell phenotype research and immune-related gene research.In order to explore the changes in the immune response of subgroup J avian leukemia virus infected memory PBMC in vitro,this experiment used surface fluorescence monoclonal antibodies of different channels to incubate with PBMC at 4 HPI and 8 HPI and detected by flow cytometry PBMC samples after antibody labeling.The results showed that compared with the control group,the percentage of CD8⁺T lymphocytes was significantly reduced at 4 HPI（p<0.05）;at 8HPI,the percentage of double positive cells by both KUL01 and MHCⅡincreased significantly（p<0.05）.In order to explore the changes in the transcription level of immune-related genes caused by ALV-J memory PBMCs,ten immune-related genes were selected for detection and analysis by q RT-PCR.The results showed that when infected with 4 HPI,the transcription levels of IL-13 and IL-4 genes were significantly increased（p<0.01）,and the transcription levels of IL-5 genes was up-regulated（p<0.05）.When infected with 8 HPI,the transcription levels of IFN-γ,IL-13,IL-4,IL-5 and Mx1 genes were significantly up-regulated（p<0.01）,and the transcription levels of CXCLi1 gene were significantly down-regulated（p<0.01）.In summary,the experiment novelly screen the model for the first time that ALV-J（SCAU-HN06 strain）infecting primary ALV-memory PBMC in vitro:after ALV-J infection without other external stimulant,when S/P≥0.2 and the number of viable cells is not less than 80%is within 8 h.Moreover,we also confirmed that ALV-J induce the expression of immune-related genes during the primary infection of primary memory PBMC in vitro,which caused a change in the percentage of T lymphocytes and antigen-presenting cells.The above results provide a potential research material and infection model for exploring the interaction between ALV and other related avian virus and the host immune system,which is helpful to further explore the pathogenic mechanism of ALV-J and provide evidence for the prevention and control of poultry industry.